Systems and methods for determining sample osmolarity

ABSTRACT

Systems and methods for determining the osmolarity of a sample are provided. Aspects of the subject methods include contacting a sensing surface of a surface plasmon resonance based sensor with a sample, and generating one or more data sets at at least two wavelengths over a time interval, wherein the data sets are used to determine the osmolarity of the sample. The subject methods find use in determining the osmolarity of a sample, such as a biological sample (e.g., a tear fluid), and in the diagnosis and/or monitoring of various diseases and disorders, such as, e.g., dry eye disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/253,595, filed on Nov. 10,2015, the disclosure of which application is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to systems and methods for determining theosmolarity of a sample, such as a biological sample (e.g., a tearfluid).

BACKGROUND OF THE INVENTION

Dry eye disease, or Keratoconjunctivitis Sicca (KCS) is one of the mostfrequently established diagnoses in ophthalmology. Current estimateshold that roughly 40-60 million people in the United States exhibit dryeye symptoms. The lack of accurate statistical data about the occurrenceof dry eye is due largely to a lack of state-of-the-art diagnosticequipment. A more disturbing trend, however, is the misdiagnosis of dryeye or its escape from early detection altogether, since symptomaticpatients are not always easily identified.

Pursuing more effective diagnosis will strengthen the paradigm ofophthalmic care, a fact recognized by the pharmaceutical industry. Thefirst prescription pharmaceuticals for treating dry eye are nowappearing on the market, with more on the way, and yet the methods fordiagnosis and monitoring treatment remain problematic.

There is no “gold standard” test that both diagnoses dry eye andmonitors the effectiveness of treatment efforts. One popular method is amatrix of subjective observation of symptoms and objective tests (suchas Schirmer testing, staining techniques and tear break-up time), noneof which is specific to the detection of dry eye or the measurement ofits severity. Considering recent pharmaceutical advancements aimed attreating dry eye, timely and parallel advancements in diagnostictechnologies are needed.

The osmolarity of a tear—the degree of dissolved solids therein—ispopularly accepted by experts in the field as an indicator of thepresence and severity of dry eye. The instrument most commonlyassociated with the measurement of tear osmolarity is the osmometer;however, technical limitations have restricted the use of tearosmometers to primarily research environments.

An osmometer is a device that measures the concentration of dissolvedsolutes in a liquid, such as water. Though it is widely used in otherfields, osmometers are used in medicine in applications such asdetermining osmol gap in toxicology and trauma cases, monitoringmannitol treatment infusions, and monitoring the absorption in glycineingestion with irrigation fluids in surgical procedures, among others.

Despite the suitability of this technology for measuring tearosmolarity, current devices present certain limitations that preventtheir widespread use in a clinical environment. The most prevalentproblem has to do with sample size.

Nearly all commercially available osmometers are designed (and perhapstechnologically limited) to measure milliliter-size samples. Tearsamples extracted from patients tend to be in the nanoliter volumes, andfurther complicating matters, dry eye patients generally have fewertears, making handling of samples even more difficult. Osmometersdesigned to measure nanoliter sample sizes are not availablecommercially and are too cumbersome for practical use in a clinicalenvironment. The result is that practicing ophthalmologists are leftwith a haphazard methodology and inadequate tools to accurately detectthis prevalent condition.

Dry eye disease is a complex group of diseases characterized by adecreased production of one or more of the three components of the tearfilm: the lipid layer, the aqueous layer, and the mucin layer. Adeficiency in one of the tear film components may lead to a loss of thetear film stability. Normal sight relies on a moist ocular surface andrequires a sufficient quality of tears, normal composition of the tearfilm, regular blinking and normal lid closure as prerequisites. If leftuntreated, dry eye syndrome can cause progressive pathological changesin the conjunctival and corneal epithelium, discomfort, cornealulceration's and even ultimately lead to blindness.

Standard treatment has been tear replacement therapy, which attempts toeither mimic the human tear film or present a more sophisticatedhypo-osmolar version of the tear film. Unfortunately, as dry eyesyndrome progresses beyond the mild stage, this common therapy becomesless effective. Further, these treatments do not address the etiology ofdry eye.

The precise mechanisms that give rise to dry eye are currently unknownand have been the subject of debate over the years. Recently, severaldifferent mechanisms have been proposed as a possible etiology of dryeye, with a general ideology that it is usually caused by a problem withthe quality of the tear film that lubricates the ocular surface. Morerecent research has proposed that dry eye may be a result of a decreasein hormonal status with aging (being more prominent in postmenopausalwomen), or have an immune basis and acquired inflammatory condition ofthe ocular surface. Other causes of dry eye symptoms can occur fromcertain medications (e.g., antihistamines, beta-blockers), associationswith certain systemic inflammatory diseases (e.g., rheumatoidarthritis), mechanical causes (e.g., incomplete closure of eyelids),infectious causes (e.g., viral infections) and certain neurologicalcauses (e.g., LASIK procedures). Despite recent gains in knowledge ofpossible pathogenic factors of dry eye, there has been a lack ofconsensus as to the appropriate diagnostic criteria, the specific aimsof objective diagnostic testing, the role subjective symptoms play indiagnosis, and the interpretation of results.

The symptoms of dry eye vary considerably from one individual toanother. Most patients complain of a foreign body sensation, burning andgeneral ocular discomfort. The discomfort is typically described as ascratchy, dry, sore, gritty, smarting or burning feeling. Discomfort isthe hallmark of dry eye because the cornea is richly supplied withsensory nerve fibers.

Despite its high prevalence, dry eye is not always easy to diagnose. Thevast majority of patients have symptoms that are mild to moderate inseverity. Although these patients are genuinely suffering discomfort,objective signs of dry eye may be missed, and without proper diagnosis,patients may not receive the attention and treatment that this conditionwarrants. The signs and symptoms of dry eye can be misinterpreted asevidence of other conditions, such as infectious, allergic, orirritative conjunctivitis. Given these complications in diagnosis, it isestimated that the diagnosis rate of dry eye is approximately 20%.

Diagnosis of dry eye typically begins with clinical examination. ASchrimer test is usually performed where standardized strips of filterpaper are placed at the junction between the middle and lateral third ofthe lower lid. If less than 5 millimeters has been wetted after 5minutes, there is reason to believe aqueous tear deficiency is present.Though the test is quick, inexpensive and results are availableimmediately, it provides only a rough estimate and is unreliable inmoderate dry eye.

Dye staining is another method of diagnosing dry eye, with eitherfluorescein or Rose Bengal, and a trained physician can look forpatterns under slit lamp observation indicating dryness. Another test,tear break-up time, is a measure of the stability of the tear film. Anormal tear film begins to break up after approximately 10 seconds, andthis time is reduced in patients with dry eye.

The osmometer generally used in measuring tear osmolarity is the CliftonDirect Reading Nanoliter Osmometer (Clifton Technical Physics, Hartford,N.Y.) developed in the 1960's. Although not necessarily originallyintended for use in measuring tears, it is one of the few instrumentscapable of measuring nanoliter volumes of solution and has found its wayinto ophthalmology.

The Clifton Osmometer was produced in limited quantities over the years,and is not routinely used outside a research laboratory. It is based onthe well-known measurement technique called freezing point depression.The Clifton Osmometer measures the osmolarity of a sample by measuringthe freezing point depression. In freezing point depressionmeasurements, water (which normally freezes at 0° C.), experiences adepression in its freezing temperature in presence of dissolved solutes,the mathematical relationship of which is defined by Raoult's Law.

Though the test can be accurate, it requires a very skilled operator tomake the measurement. The test monitors the depression in freezingtemperature by examining a fractional volume of a teardrop under amicroscope. Due to its limitations and lack of availability, thereappears to be only a few units left in the field. Furthermore eachmeasurement can take over fifteen minutes, which, coupled with the smallsample volumes, make the use of the Clifton Osmometer an extremelytedious and inconvenient process. The amount of time required and theoperating skill demanded are unacceptable to a busy practice or clinic,even if the units were available.

There is a need for simple and accurate sensors, systems and methodsthat can determine the osmolarity of a sample, such as, e.g., abiological sample, e.g., a tear fluid, and to use the osmolarity data todiagnose and/or monitor treatment efforts for various diseases anddisorders, such as, e.g., dry eye disease.

SUMMARY

Systems and methods for determining the osmolarity of a sample areprovided. Aspects of the subject methods include contacting a sensingsurface of a sensor with a sample, and generating one or more data setsover a time interval, wherein the data sets are used to determine theosmolarity of the sample. The subject methods find use in determiningthe osmolarity of a sample, such as a biological sample (e.g., a tearfluid), and in the diagnosis and/or monitoring of various diseases anddisorders, such as, e.g., dry eye disease.

Aspects of the invention include systems comprising: (i) a sensorcomprising a sensing surface comprising a coated region, wherein thesensor is configured to: direct a first optical signal to interact withthe sensing surface at a first incident angle; and direct a secondoptical signal to interact with the sensing surface at a second incidentangle; and (ii) an optical chassis comprising: an optical signalgenerating component; a detection component; a processor; a controller;and a computer-readable medium comprising instructions that, whenexecuted by the processor, cause the controller to: direct an opticalsignal having a first wavelength to interact with the sensing surface atthe first incident angle to generate a first surface plasmon resonance(SPR) signal; generate a series of images of the first SPR signal over afirst time interval using the detection component; determine a series ofpixel positions that correspond to a minimum value of the first SPRsignal over the first time interval; direct an optical signal having asecond wavelength to interact with the sensing surface at the firstincident angle to generate a second SPR signal; generate a series ofimages of the second SPR signal over a second time interval using thedetection component; determine a series of pixel positions thatcorrespond to a minimum value of the second SPR signal over the secondtime interval; compare the series of pixel positions that correspond tothe minimum value of the first SPR signal over the first time intervalto the pixel position of at least one reference feature to generate afirst reference-corrected SPR function; compare the series of pixelpositions that correspond to the minimum value of the second SPR signalover the second time interval to the pixel position of the at least onereference feature to generate a second reference-corrected SPR function;and compare one or more characteristics of the first reference-correctedSPR function and the second reference-corrected SPR function todetermine a reference-corrected SPR delta pixel value.

In some embodiments, the first incident angle ranges from about 40 toabout 70 degrees. In some embodiments, the first incident angle rangesfrom about 62 to about 67 degrees. In some embodiments, the firstincident angle is about 64 degrees. In some embodiments, thecomputer-readable medium further comprises instructions that, whenexecuted by the processor, cause the controller to compare thereference-corrected SPR delta pixel value to a calibration data set.

In some embodiments, the computer-readable medium further comprisesinstructions that, when executed by the processor, cause the controllerto: direct an optical signal having a first wavelength to interact withthe sensing surface at a second incident angle to generate a thirdsurface plasmon resonance (SPR) signal; generate an image of the thirdSPR signal using the detection component; determine a pixel position ofa minimum value of the third SPR signal on the generated image; directan optical signal having a second wavelength to interact with thesensing surface at the second incident angle to generate a fourth SPRsignal; generate an image of the fourth SPR signal using the detectioncomponent; determine a pixel position of a minimum value of the fourthSPR signal on the generated image; and compare the pixel position of theminimum value of the third SPR signal to the pixel position of theminimum value of the fourth SPR signal to determine an SPR delta pixelvalue.

In some embodiments, the second incident angle ranges from about 40 toabout 70 degrees. In some embodiments, the second incident angle rangesfrom about 40 to about 45 degrees. In some embodiments, the secondincident angle is about 42 degrees.

In some embodiments, the computer-readable medium further comprisesinstructions that, when executed by the processor, cause the controllerto compare the SPR delta pixel value to a calibration data set.

In some embodiments, the computer-readable medium further comprisesinstructions that, when executed by the processor, cause the controllerto: direct the optical signal having the first wavelength to interactwith the sensing surface at the second incident angle to generate afirst critical angle signal; generate an image of the first criticalangle signal using the detection component; determine a pixel positionof a maximum value of the first critical angle signal on the generatedimage; direct an optical signal having a second wavelength to interactwith the sensing surface at the first incident angle to generate asecond critical angle signal; generate an image of the second criticalangle signal using the detection component; determine a pixel positionof a maximum value of the second critical angle signal on the generatedimage; and compare the pixel position of the maximum values of first andsecond critical angle signals to determine a critical angle delta pixelvalue.

In some embodiments, the sensor comprises a coated region and anon-coated region, and wherein the first and second critical anglesignals are generated from the non-coated region. In some embodiments,the reference feature comprises a pixel position of one or moreopto-mechanical reference features. In some embodiments, the referencefeature comprises the pixel position of the minimum value of the thirdSPR signal, the pixel position of the minimum value of the fourth SPRsignal, the SPR delta pixel value, or a combination thereof. In someembodiments, the reference feature comprises the pixel position of themaximum value of the first critical angle signal, the pixel position ofthe maximum value of the second critical angle signal, the criticalangle delta pixel value, or a combination thereof. In some embodiments,the characteristic of the first and second reference-corrected SPRfunctions comprises a derivative of the first and secondreference-corrected SPR functions. In some embodiments, thecharacteristic of the first and second reference-corrected SPR functionscomprises a plateau value of the first and second reference-correctedSPR functions. In some embodiments, the sensor is configured to beremovably coupled to the optical chassis. In some embodiments, thesystem is a benchtop system. In some embodiments, the system is ahand-held system.

Aspects of the invention include methods for determining the osmolarityof a sample, the method comprising: contacting a sensing surface of asystem as described herein with the sample; directing the optical signalhaving the first wavelength to interact with the sensing surface at thefirst incident angle to generate a first surface plasmon resonance (SPR)signal; generating a series of images of the first SPR signal over afirst time interval using the detection component; determining a seriesof pixel positions that correspond to a minimum value of the first SPRsignal over the first time interval; directing the optical signal havingthe second wavelength to interact with the sensing surface at the firstincident angle to generate a second SPR signal; generating a series ofimages of the second SPR signal over a second time interval using thedetection component; determining a series of pixel positions thatcorrespond to a minimum value of the second SPR signal over the secondtime interval; comparing the series of pixel positions that correspondto the minimum value of the first SPR signal over the first timeinterval to the pixel position of at least one reference feature togenerate a first reference-corrected SPR function; comparing the seriesof pixel positions that correspond to the minimum value of the secondSPR signal over the second time interval to the pixel position of the atleast one reference feature to generate a second reference-corrected SPRfunction; comparing one or more characteristics of the firstreference-corrected SPR function and the second reference-corrected SPRfunction to determine a reference-corrected SPR delta pixel value; andcomparing the reference-corrected SPR delta pixel value to a calibrationdata set to determine the osmolarity of the sample.

In some embodiments, the methods further comprise: contacting thesensing surface with a reference medium; directing the optical signalhaving the first wavelength to interact with the sensing surface at thesecond incident angle to generate a third surface plasmon resonance(SPR) signal; generating an image of the third SPR signal using thedetection component; determining a pixel position of a minimum value ofthe third SPR signal on the generated image; directing the opticalsignal having the second wavelength to interact with the sensing surfaceat the second incident angle to generate a fourth SPR signal; generatingan image of the fourth SPR signal using the detection component;determining a pixel position of a minimum value of the fourth SPR signalon the generated image; and comparing the pixel position of the minimumvalue of the third SPR signal to the pixel position of the minimum valueof the fourth SPR signal to determine an SPR delta pixel value.

In some embodiments, the methods further comprise comparing the SPRdelta pixel value to the reference-corrected SPR delta pixel value. Insome embodiments, the methods further comprise comparing the SPR deltapixel value to a calibration data set. In some embodiments, the methodsfurther comprise: directing the optical signal having the firstwavelength to interact with the sensing surface at the second incidentangle to generate a first critical angle signal; generating an image ofthe first critical angle signal using the detection component;determining a pixel position of a maximum value of the first criticalangle signal on the generated image; directing the optical signal havingthe second wavelength to interact with the sensing surface at the secondincident angle to generate a second critical angle signal; generating animage of the second critical angle signal using the detection component;determining a pixel position of a maximum value of the second criticalangle signal on the generated image; and comparing the pixel position ofthe maximum values of first and second critical angle signals todetermine a critical angle delta pixel value. In some embodiments, themethods further comprise comparing the critical angle delta pixel valueto the reference-corrected SPR delta pixel value. In some embodiments,the methods further comprise comparing the critical angle delta pixelvalue to the SPR delta pixel value. In some embodiments, the methodsfurther comprise comparing the critical angle delta pixel value to acalibration data set.

In some embodiments, the images of the SPR signals are captured in asingle image frame. In some embodiments, the images of the SPR signalsand the images of the critical angle signals are captured in a singleimage frame.

In some embodiments, the methods further comprise: comparing thereference-corrected SPR delta pixel value, the SPR delta pixel value, orthe critical angle delta pixel value to an external environmentparameter to generate an external environment corrected delta pixelvalue; and comparing the external environment corrected delta pixelvalue to a calibration data set. In some embodiments, the externalenvironment parameter is selected from the group comprising:temperature, pressure, humidity, light, environmental composition, orany combination thereof.

In some embodiments, the optical signals having a first and a secondwavelength are directed to interact with the sensing surfacesimultaneously. In some embodiments, the optical signals having a firstand second wavelength are directed to interact with the sensing surfacein a gated manner. In some embodiments, the calibration data set isstored in a read-only memory of a processor of the system.

In some embodiments, the sample is a biological sample. In someembodiments, the biological sample is a tear fluid. In some embodiments,the reference medium is air.

In some embodiments, the first time interval ranges from about 0.001seconds to about 90 seconds. In some embodiments, the second timeinterval ranges from about 0.001 seconds to about 90 seconds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a relationship between tear osmolarity andprobability for normal eyes and dry eyes.

FIG. 2, Panel A is an illustration demonstrating the Surface PlasmonResonance (SPR) technique for measuring the osmolarity of a tear fluid.Panel B is a graph showing relative response as a function of SPR angle.

FIG. 3, Panel A is an image generated using a 638 nm wavelength laser,and Panel B is an image generated using a 632 nm wavelength traditionalLED. Panel C is a graph showing a larger amount of noise from the laserdiode image. Panel D is a graph showing a lower amount of noise from theLED. The graph in Panel D is noticeably smoother than the graph in PanelC.

FIG. 4 is a graph comparing percent reflectivity as a function of angleof incidence for three different optical signals that have differentwavelengths. The longer wavelength optical signals have narrower(sharper) SPR line widths.

FIG. 5 is a collection of three different images demonstrating thedifference in image quality for different optical sources with differentwavelengths. The width of the SPR line is narrower for light having alarger wavelength.

FIG. 6 is a graph showing resolution as a function of wavelength for ahigh index of refraction glass (SF10, refractive index˜1.72) and a lowerindex of refraction glass (BK7, refractive index˜1.52). The graph showsthat there is little difference between the different materials.

FIG. 7 is a graph demonstrating the straight line fit approach fordetermining a minimum value of an SPR curve.

FIG. 8 is an SPR line image acquired using a video imager. A region ofinterest within the image is outlined with the depicted rectangle.

FIG. 9 is a graph showing the gray scale value as a function of pixelposition for the region of interest depicted in FIG. 8. The graph wasgenerated corresponding to the average of the vertical column pixelintensity in the region of interest along the X direction.

FIG. 10 is a graph showing the SPR curve depicted in FIG. 9 (dottedline) as well as the derivative of the SPR curve (solid line) as afunction of SPR angle (pixels). The zero crossing of the derivative ofthe SPR curve is circled.

FIG. 11 is a graph showing the location of the zero crossing of thederivative of the SPR curve depicted in FIG. 10 to a fraction of a pixelvalue.

FIG. 12 is a graph showing determination of the exact coordinate of thezero crossing point using a linear interpolation technique.

FIG. 13 is a table showing the location of SPR minima for 10 SPR imagessequentially acquired at approximately 1.0 second intervals.

FIG. 14 is a graph showing a relative SPR response for ethanol and fordeionized water. The difference in pixel position for the two media isshown as approximately 910 pixels.

FIG. 15 is an image showing raw SPR data for an ethanol solution.

FIG. 16 is an image showing raw SPR data for an deionized watersolution.

FIG. 17 is a graph showing osmolarity as a function of SPR angle(pixels) acquired and analyzed using the derivative signal processingtechnique.

FIG. 18 is a graph showing relative response as a function of pixelcount that was generated using a curve fitting technique.

FIG. 19 is a graph showing relative response as a function of pixelcount that was generated by fitting a cubic polynomial to the SPR curve.

FIG. 20 shows quadratic and cubic equation solutions that can be used todetermine the pixel position corresponding to SPR minimum value.

FIG. 21 is a graph showing the relative change of the index ofrefraction with temperature for a variety of example materials.

FIG. 22 is an illustration of an example of an injection molded sensor.The sensor and the sensing surface are referenced.

FIG. 23 is an illustration of another example of an injection moldedsensor.

FIG. 24 is an illustration of another example of an injection moldedsensor. The depicted sensor is configured to direct a first opticalsignal to interact with a sensing surface at an incident angle of 42.04degrees, and to direct a second optical signal to interact with thesensing surface at an incident angle of 64.44 degrees.

FIG. 25 is an illustration of another example of an injection moldedsensor. The depicted sensor is configured to direct a first opticalsignal to interact with a sensing surface at an incident angle of 42.04degrees, and to direct a second optical signal to interact with thesensing surface at an incident angle of 64.44 degrees.

FIG. 26 is an illustration showing various light paths moving through aplurality of optical chassis components and a sensor.

FIG. 27 is another illustration showing various light paths movingthrough a plurality of optical chassis components and a sensor.

FIG. 28, Panel A is another illustration showing various light pathsmoving through a plurality of optical chassis components and a sensor.Panel B shows an end view of a sensing surface, showing a coated regionand a non-coated region. Panel C is a close-up illustration of variouslight paths interacting with various facets and a sensing surface of asensor.

FIG. 29, Panel A shows a simulation of an air SPR line (obtained from acoated region of the sensing surface) and the critical angle transition(obtained from a non-coated region of the sensing surface) using one LEDfrom a first set of LEDs from a dry sensing surface (in contact withair). Panel B illustrates an SPR line obtained using one LED from asecond set of LEDs when the sensing surface has been contacted withwater or tear fluid.

FIG. 30 illustrates the geometry of Snell's Law (the law of refraction)and the critical angle of a substrate.

FIG. 31 is a graph of reflectance as a function of angle of incidencefor a plurality of sensing surfaces having different thicknesses of goldfilm. The critical angle (θ_(c)) remains constant, and is independent ofthe thickness of the gold film.

FIG. 32, Panel A is another illustration showing various light pathsmoving through a plurality of optical chassis components and a sensor.Panel B is a close-up illustration of various light paths interactingwith various facets and a sensing surface of a sensor.

FIG. 33, Panel A is a simulated image showing data from a tear fluidsample. The air SPR line and tear SPR line are shown, as well as thecritical angle line. Panel B is a graph showing gray-scale value as afunction of pixel position for the image in Panel A. The minimumgray-scale value corresponding to the air and tear SPR lines are shown,as well as the maximum gray-scale value corresponding to the criticalangle line.

FIG. 34 is another illustration showing various light paths movingthrough a plurality of optical chassis components and a sensor.

FIG. 35 is another illustration showing various light paths movingthrough a plurality of optical chassis components and a sensor.

FIG. 36 is another illustration showing various light paths movingthrough a plurality of optical chassis components and a sensor. Theoverall length of the depicted optical chassis is 2.181 inches.

FIG. 37 is a side view illustration of an optical chassis and a sensor.The overall height of the depicted optical chassis is 0.903 inches. Thediameter of the depicted sensor is 0.765 inches.

FIG. 38 is another side view illustration of an optical chassis and asensor.

FIG. 39 is another side view illustration of an optical chassis and asensor.

FIG. 40 is a perspective illustration of an optical chassis and asensor.

FIG. 41 is another side view illustration of an optical chassis and asensor.

FIG. 42, Panel A is a side view illustration of a sensor. Panel B is abottom view illustration of a sensor.

FIG. 43 is a perspective illustration of a sensor.

FIG. 44, Panels A and B show side view illustrations of a sensor.

FIG. 45 is and end view illustration of a sensor.

FIG. 46 is and end view illustration of a sensor and an optical chassis.

FIG. 47 is a transparent rendering of a sensor.

FIG. 48 is an illustration of a benchtop system comprising a sensor andan optical chassis comprising various components.

FIG. 49 is a perspective illustration of a benchtop system.

FIG. 50 is another perspective illustration of a benchtop system.

FIG. 51 is an image of an outer casing component that can be used inconjunction with a benchtop system as illustrated in FIGS. 48-50.

FIG. 52, Panels A-E show images and graphs of SPR signals collected overdifferent time intervals using the methods described herein.

FIG. 53 Panels A-D show images and graphs of SPR signals collected overdifferent time intervals using the methods described herein.

FIG. 54 is a graph showing delta pixel value as a function of time fortwo different SPR signals that were obtained from a sample of tear fluidhaving an osmolarity of 300 mOsm/L.

FIG. 55 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is80% of the protein content of normal tears, using the methods describedherein.

FIG. 56 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is80% of the protein content of normal tears, using the methods describedherein.

FIG. 57 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is120% of the protein content of normal tears, using the methods describedherein.

FIG. 58 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is120% of the protein content of normal tears, using the methods describedherein.

FIG. 59 is a set of graphs showing the results from a comparativeanalysis of a sample of tear fluid having a protein content that is 80%of the protein content of normal tears, and a sample of tear fluidhaving a protein content that is 120% of the protein content of normaltears, using the subject methods.

FIG. 60 is a set of graphs showing the results from a comparativeanalysis of a normal tear fluid sample using the subject methods.

FIG. 61 is a graph showing the delta pixel value of a salt solution as afunction of osmolarity, analyzed using two different wavelengths.

DETAILED DESCRIPTION

Systems and methods for determining the osmolarity of a sample areprovided. Aspects of the subject methods include contacting a sensingsurface of a sensor with a sample, and generating one or more data setsover a time interval, wherein the data sets are used to determine theosmolarity of the sample. The subject methods find use in determiningthe osmolarity of a sample, such as a biological sample (e.g., a tearfluid), and in the diagnosis and/or monitoring of various diseases anddisorders, such as, e.g., dry eye disease.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular aspectsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular aspects only, and is not intended to be limiting, since thescope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating un-recited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual aspects described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalaspects without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

The term “sensing surface” as used herein refers to a surface of asensor that is configured to contact an external medium.

The terms “incident angle” or “angle of incidence” as usedinterchangeably herein refer to an angle that is formed between a beamof light that is directed toward a planar surface, and a line that isperpendicular to the same planar surface.

The term “facet” as used herein refers to a substantially planar portionof a surface (e.g., an interior surface or an exterior surface) of asensor.

The term “semitransparent film” as used herein refers to a film that ispartially transparent to light and facilitates surface plasmon/polaritongeneration.

The terms “reflective coating” and “reflective film”, as usedinterchangeably herein, refer to a coating or a film, respectively, thatare capable of reflecting light or other radiation. The terms“semitransparent film” and “reflective film” or “reflective coating” asused herein are not mutually exclusive, and a given film can be both asemitransparent film as well as a reflective film.

The team “noble metal” as used herein refers to a metallic element thatis resistant to corrosion in moist air. Non-limiting examples of noblemetals include Copper (Cu), Ruthenium (Ru), Rhodium (Rh), Palladium(Pd), Silver (Ag), Rhenium (Re), Osmium (Os), Iridium (Ir), Platinum(Pt), Gold (Au), Mercury (Hg), or combinations thereof.

The term “adhesion layer” as used herein refers to a layer of materialthat is formed on a sensing surface or on a facet, and which facilitatesadhesion of a coating material (e.g., a reflective film or asemitransparent film) to the sensing surface or facet.

The term “coated region” as used herein with reference to a sensingsurface or facet means a region of the sensing surface or facet that iscovered with a coating (e.g., a semitransparent film, a reflectivecoating, and/or an adhesion layer). The term “non-coated region” as usedherein with reference to a sensing surface or facet means a region ofthe sensing surface or facet that is not covered with a coating.

The term “optical chassis” as used herein refers to a structure thatsupports and/or contains one or more optical components.

The term “optical signal” as used herein refers to a signal thatcomprises photons.

The term “critical angle” as used herein refers to an angle of incidenceabove which (e.g., at an angle of incidence having a larger angularvalue than the critical angle) total internal reflection occurs.

The term “pixel position” as used herein refers to the position of apixel on a coordinate system, such as, e.g., an x,y coordinate plane.

The term “compare” as used herein with respect to comparing pixelpositions refers to measuring a difference in position of two or morepixels on a coordinate plane. Comparing of pixel positions can bequalitative or quantitative.

The term “reference feature” as used herein refers to one or more datapoints that do not vary with time, or a component that is configured oradapted to generate one or more data points that do not vary with time.

The term “opto-mechanical reference” or “OMR” refers to a component thatis configured or adapted to place a physical obstruction in the path ofone or more optical signals and to thereby generate one or morereference signals that do not vary with time, and that can be detectedand analyzed by a detection component.

The terms “delta pixel position” or “delta pixel value” as used hereinrefer to a numerical value that represents a difference in positionbetween two pixels on a coordinate system.

The term “external environment parameter” as used herein refers to acharacteristic of an environment that is external to a subject sensor orsystem. A non-limiting example of an external environment parameter isthe temperature of a room in which a sensor is operated.

The term “corrected” as used herein with respect to a data value refersto a data value that has undergone a mathematical manipulation, e.g.,has been multiplied or divided by a numerical value to correct ornormalize the data value based on a given parameter (e.g., an externalenvironment parameter, or a reference value).

The term “reference-corrected” as used herein with respect to a datavalue or a mathematical function (e.g., an SPR function) refers to adata value or mathematical function that has undergone a mathematicalmanipulation, e.g., has been multiplied or divided by at least onenumerical value obtained from one or more reference features to corrector normalize the data value based on the at least one numerical valueobtained from the reference feature.

The term “calibration data set” as used herein refers to a collection ofone or more data points that represent a relationship between ameasurement standard and a characteristic that is measured by a subjectsensor and/or system.

The term “function” as used herein refers to a mathematical operation,or graphical representation thereof, wherein a unique y coordinate valueis assigned to every x coordinate value.

The term “minimum value” as used herein refers to the lowest numericalvalue of a function in an image frame and on a given coordinate system.

The term “maximum value” as used herein refers to the highest numericalvalue of a function in an image frame and on a given coordinate system.

The term “derivative” as used herein refers to a rate of change of afunction. The value of a derivative of a function is the slope of thetangent line at a point on a graph representing the function.

The term “plateau value” as used herein refers to a y-value of afunction over a region where the function has a substantially constant,or steady-state, y-value.

The term “quality parameter” as used herein refers to an aspect of asubject sensor or system that is required for optimal functioning of thesensor or system.

The term “surface plasmon resonance” or “SPR” as used herein refers to aresonant oscillation of conduction electrons at an interface between anegative and a positive permittivity material that is stimulated byincident light.

The term “optical signal manipulation component” as used herein refersto a component that is capable of manipulating one or more features ofan optical signal. An optical signal manipulation component can includeany number of individual components, which individual components can actin parallel and/or in series to manipulate one or more characteristicsof an optical signal. Non-limiting examples of optical signalmanipulation components include: beam splitters, spatial filters,filters that reduce external ambient light, lenses, polarizers, andoptical waveguides.

The term “removably couple” as used herein refers to connecting two ormore components in such a way that the connection is reversible, and thecomponents can be separated from one another.

The term “retention component” as used herein refers to a component thatis configured to retain one or more components in a fixed position withrespect to another component.

The term “alignment component” as used herein refers to a component thatis configured to provide functional and/or structural alignment betweentwo or more components that are operably coupled.

The term “kinematic mounting component” as used herein refers to amounting component that provides a number of constraints that is equalto the number of degrees of freedom in the component being mounted.

The term “benchtop system” as used herein refers to a system that isconfigured to be disposed on a surface of, e.g., a laboratory benchtop,or another suitable substrate, during operation.

The term “hand-held system” as used herein refers to a system, or acomponent thereof, that is configured to be held in a user's hand duringoperation.

The terms “subject” or “patient” as used herein refer to any human ornon-human animal.

Sensors and Systems

Aspects of the invention include sensors and systems configured to carryout the subject methods, e.g., to determine the osmolarity of a sample.In certain embodiments, the subject systems include an optical sensorhaving at least one sensing surface and configured to direct a firstoptical signal to interact with the sensing surface at a first incidentangle, and to direct a second optical signal to interact with thesensing surface at a second incident angle. In some embodiments, thesubject systems further include an optical chassis that includes anoptical signal generation component and a detection component. Each ofthese components is now further described in greater detail.

Sensors

As summarized above, aspects of the invention include sensors thatinclude at least one sensing surface, and that are configured to directa first optical signal to interact with the sensing surface at a firstincident angle, and to direct a second optical signal to interact withthe sensing surface at a second incident angle. By directing opticalsignals to interact with the sensing surface at two different incidentangles, the subject sensors are capable of generating data from thesensing surface for two or more different media (e.g., air and water),and detecting the data using the same detection component. As such, dataobtained from different media can be captured in the same field of view,or image frame, of a detection component, and can then be analyzed bythe detection component. Analysis of the data can then be used todetermine one or more characteristics of the media. The inclusion ofdata from the sensing surface for different media in the same field ofview, or image frame, of the detection component provides an internalreference within the data that can be used in analysis (e.g., can beused for calibration of the sensor and/or for analyzing an unknownsample). As described further herein, in some embodiments, a sensor caninclude a reference feature that can be used in data analysis. In someembodiments, a sensor comprises a reference feature that creates areference signal in an image frame of the detection component, and oneor more pixel positions of the reference signal can be used as aninternal reference for purposes of data analysis (e.g., can be used forcalibration of the sensor and/or for analyzing a known or unknownsample).

The subject sensors include at least one sensing surface that comprisesa semitransparent film, wherein the semitransparent film comprises anoble metal. The semitransparent film facilitates surface plasmonresonance (SPR)-based analysis of a medium in contact with the sensingsurface. SPR is a phenomenon that occurs when light is incident on asensing surface at a particular angle, so that the reflected light isextinguished. At a particular angle of incident light, the intensity ofthe reflected light shows a characteristic curve of diminishingintensity, well defined by mathematical equations. The angle of incidentlight that corresponds to a reflectivity minimum of the curve isinfluenced by the characteristics of the semitransparent film and theexternal medium that is in contact therewith. FIG. 2, Panel A providesan illustrative overview of the SPR technique for tear osmolaritymeasurement. FIG. 2, Panel B provides a graph of an SPR signal (i.e., anSPR signal curve, or function), demonstrating the relative minimum ofthe SPR curve, and indicating the position corresponding to areflectivity minimum of the SPR signal curve. In some embodiments,aspects of the invention include determining a pixel positioncorresponding to a reflectivity minimum of an SPR signal curverepresented on an image that is generated by a detection component(described further herein).

In some embodiments, the semitransparent film on the sensing surface canrange in thickness from about 0.5 nm up to about 200 nm, such as about 1nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 41 nm, 42 nm,43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53nm, 54 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 100nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190nm, or 195 nm. A semitransparent film can be deposited on a surface of asensor using any suitable technique, for example, thin film depositiontechniques (e.g., atomic layer deposition (ALD), chemical vapordeposition (CVD), evaporative deposition, metal organic chemical vapordeposition (MOCVD), sputtering, etc.), or any combination thereof.Non-limiting examples of noble metals that can be used in asemitransparent film in accordance with embodiments of the subjectsensors include Copper (Cu), Ruthenium (Ru), Rhodium (Rh), Palladium(Pd), Silver (Ag), Rhenium (Re), Osmium (Os), Iridium (Ir), Platinum(Pt), Gold (Au), Mercury (Hg), or any combination thereof. In someembodiments, a semitransparent film on a sensing surface can be composedof a plurality of discrete layers of material, wherein the material ineach layer can be selected from the noble metals described above, or anycombination thereof (e.g., alloys thereof, such as alloys of 2, 3, 4, 5,6, 7, or 8 or more different noble metals). In some embodiments, asensing surface can comprise a substrate, such as, e.g., a microscopeslide, having one side that is at least partially coated with asemitransparent film. In such embodiments, the substrate can be operablycoupled to the sensor to provide a sensing surface.

In some embodiments, a sensor can include an adhesion layer that isdeposited on a sensing surface between the sensor (or substrate) and asemitransparent film. An adhesion layer in accordance with embodimentsof the invention serves to promote adhesion of the semitransparent filmto the sensing surface, and can modulate one or more properties of anoptical signal passing through the sensor. For example, in someembodiments, an adhesion layer can comprise a material that improves adesired property of an optical signal that passes through the adhesionlayer. In some embodiments, the thickness and material composition of anadhesion layer are selected to favorably manipulate a property of anoptical signal that passes through the adhesion layer. In someembodiments, a material having a desired refractive index (RI) isselected to modulate a characteristic of an optical signal that passesthrough the adhesion layer. In some embodiments, the adhesion layercomprises a material that modulates a characteristic of an opticalsignal passing therethrough, e.g., reduces the amount of noise in theoptical signal.

In some embodiments, an adhesion layer can range in thickness from about0.5 nm up to about 200 nm, such as about 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185nm, 190 nm, or 195 nm. An adhesion layer can be deposited on a surfaceof the sensor using any suitable technique, for example, thin filmdeposition techniques (e.g., atomic layer deposition (ALD), chemicalvapor deposition (CVD), evaporative deposition, metal organic chemicalvapor deposition (MOCVD), sputtering, etc.), or any combination thereof.Non-limiting examples of materials that can be used in an adhesion layerin accordance with embodiments of the subject sensors include Chromium(Cr), TiO₂, TO_(x), SiO₂, SiO_(x), or any combination thereof (e.g.,mixtures or alloys thereof).

Sensing surfaces in accordance with embodiments of the invention canhave any suitable size and shape. In some embodiments, a sensing surfacecan be square, rectangular, trapezoidal, octagonal, elliptical, orcircular in shape, or any combination thereof. The surface area of asensing surface can vary, and in some embodiments can range from about 1mm² up to about 10 mm², such as about 2 mm², 3 mm², 4 mm², 5 mm², 6 mm²,7 mm², 8 mm², or 9 mm².

In certain embodiments, a sensing surface can comprise a coated regionand a non-coated region. In some embodiments, a coated region comprisesa percentage of the area of the sensing surface that ranges from about10% up to 100%, such as about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the area of thesensing surface. In certain embodiments, an entire sensing surface iscoated with a semitransparent film.

A coated region in accordance with embodiments of the invention can haveany suitable shape. In some embodiments, a coated region of a sensingsurface can be square, rectangular, trapezoidal, octagonal, elliptical,or circular in shape, or any combination thereof. In some embodiments, asensing surface can comprise a plurality of discrete coated regions,such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 discrete coated regions. A coatedregion of a sensing surface can be located in any suitable position on asensing surface. For example, in some embodiments, a coated region canbe centered on a sensing surface, while in some embodiments, a coatedregion can be, e.g., located on one particular side of a sensingsurface, located along one or more sides of a sensing surface, or thelike. In some embodiments, approximately half of the sensing surfacecomprises a coated region, while approximately half of the sensingsurface comprises a non-coated region. In some embodiments,approximately two thirds (approximately 66%) of the sensing surfacecomprises a coated region, while approximately one third (approximately33%) of the sensing surface comprises a non-coated region. In certainembodiments, the entire surface of a sensing surface is a coated region(i.e., 100% of the sensing surface is coated with a semitransparentfilm).

In some embodiments, a non-coated region of a sensing surfacefacilitates analysis of a critical angle associated with the sensor. Thecritical angle is the incident angle above which total internalreflection occurs. The critical angle is influenced by thecharacteristics of the material from which the sensor is made, and isnot influenced by the external medium that is in contact with a sensingsurface of the sensor. As such, the critical angle for a given sensorcan serve as an internal reference during analysis. In some embodiments,aspects of the invention include determining a critical angle for asensor, as well as determining a pixel position corresponding to thecritical angle on an image that is generated by a detection component(described further herein).

Sensors in accordance with embodiments of the invention can have anysuitable size and shape. In some embodiments, a sensor has ahemi-cylinder shape, having a planar surface and a curved surface,wherein the sensing surface is disposed on the planar surface. In someembodiments, a sensor comprises a conical or frustoconical shape. Insome embodiments, a sensor can have a concave shape, such that thesensor comprises an interior surface (e.g., a surface inside theconcavity) and an exterior surface. In some embodiments, a sensor canhave a frustoconical, concave shape.

In some embodiments, a sensor can have a length dimension that rangesfrom about 1 to about 20 cm, such as 2 cm, 3 cm, 4 cm, 5 cm, 8 cm, 10cm, 12 cm, 14 cm, 16 cm, or 18 cm. In some embodiments, a sensor canhave a width dimension that ranges from about 1 to about 20 cm, such as2 cm, 3 cm, 4 cm, 5 cm, 8 cm, 10 cm, 12 cm, 14 cm, 16 cm, or 18 cm. Insome embodiments, a sensor can have a height dimension that ranges fromabout 1 to about 20 cm, such as 2 cm, 3 cm, 4 cm, 5 cm, 8 cm, 10 cm, 12cm, 14 cm, 16 cm, or 18 cm. In some embodiments, a sensor can have adiameter that ranges from about 1 to about 20 cm, such as 2 cm, 3 cm, 4cm, 5 cm, 8 cm, 10 cm, 12 cm, 14 cm, 16 cm, or 18 cm.

In some embodiments, a sensor can comprise one or more facets that areconfigured to direct an optical signal in a given direction (e.g., toreflect off the facet at a given angle). Facets in accordance withembodiments of the invention can have any suitable area, and in someembodiments can range in area from about 1 mm² up to about 100 mm², suchas about 5 mm², 10 mm², 15 mm², 20 mm², 25 mm², 30 mm², 35 mm², 40 mm²,45 mm², 50 mm², 55 mm², 60 mm², 65 mm², 70 mm², 75 mm², 80 mm², 85 mm²,90 mm², or 95 mm². Facets in accordance with embodiments of the sensorcan have any suitable shape, and in some embodiments can be square,rectangular, trapezoidal, octagonal, elliptical, or circular in shape,or any combination thereof.

Sensors in accordance with embodiments of the invention can have anysuitable number of facets on a given surface of the sensor. For example,in some embodiments, a sensor can have a number of facets ranging from 1up to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9 facets on a given surface ofthe sensor. In certain embodiments, a sensor can have one or more facetson an internal surface, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 facetson an internal surface, and can also have one or more facets on anexternal surface, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 facets on anexternal surface. In some embodiments, a facet can be coated with anoptically reflective material to enhance the ability of the facet toreflect an optical signal. In some embodiments, a plurality of facetscan have a different shape and/or area. In some embodiments, a pluralityof facets can have the same shape and/or area.

In certain embodiments, one or more facets can be coated with areflective coating (e.g., a reflective film, or an optically reflectivematerial). In some embodiments, all of the facets of a sensor can becoated with a reflective coating. In some embodiments, certain facets ona sensor are coated with a reflective coating, whereas other facets onthe same sensor are not coated with a reflective coating. In someembodiments, the entire surface of a selected facet can be coated with areflective coating. In some embodiments, only a portion or section ofthe surface of a particular facet is coated with a reflective coating.In a preferred embodiment, a plurality of “shoulder” facets are coatedwith a reflective gold coating. For example, in one preferredembodiment, the facets that are labeled in FIG. 43 (as well as thosethat are symmetrically located on the opposite side of the sensingsurface) are coated with a reflective coating (e.g., a reflective goldcoating).

In some embodiments, a reflective coating on the surface of a facet canrange in thickness from about 0.1 nm up to about 1,000 nm (1 μm), suchas about 0.5 nm, about 1 nm, about 5 nm, about 10 nm, about 20 nm, about30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm,about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm,about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm,about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm,about 800 nm, about 850 nm, about 900 nm, or about 950 nm or more. Areflective coating can be deposited on a surface of a facet using anysuitable technique, such as, for example, thin film depositiontechniques (e.g., atomic layer deposition (ALD), chemical vapordeposition (CVD), evaporative deposition, metal organic chemical vapordeposition (MOCVD), sputtering, etc.), or any combination thereof.Non-limiting examples of noble metals that can be used in a reflectivefilm in accordance with embodiments of the subject sensors includeCopper (Cu), Ruthenium (Ru), Rhodium (Rh), Palladium (Pd), Silver (Ag),Rhenium (Re), Osmium (Os), Iridium (Ir), Platinum (Pt), Gold (Au),Mercury (Hg), or any combination thereof. In a preferred embodiment, areflective coating comprises gold (Au).

In some embodiments, a sensor can include an adhesion layer that isdeposited on one or more facets and is positioned between the sensor (orsubstrate) and a reflective coating on the facet. An adhesion layer inaccordance with embodiments of the invention serves to promote adhesionof the reflective coating to the facet, and can modulate one or moreproperties of an optical signal that is reflected off the facet. Forexample, in some embodiments, an adhesion layer can comprise a materialthat improves a desired property of an optical signal that is reflectedoff a particular facet. In some embodiments, the thickness and materialcomposition of an adhesion layer are selected to favorably manipulate aproperty of an optical signal that is reflected off a particular facet.

In some embodiments, an adhesion layer can range in thickness from about0.5 nm up to about 200 nm, such as about 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185nm, 190 nm, or 195 nm. An adhesion layer can be deposited on a surfaceof the sensor (e.g., on a facet of the sensor) using any suitabletechnique, for example, thin film deposition techniques (e.g., atomiclayer deposition (ALD), chemical vapor deposition (CVD), evaporativedeposition, metal organic chemical vapor deposition (MOCVD), sputtering,etc.), or any combination thereof. Non-limiting examples of materialsthat can be used in an adhesion layer in accordance with embodiments ofthe subject sensors include Chromium (Cr), TiO₂, TO_(x), SiO₂, SiO_(x),or any combination thereof (e.g., mixtures or alloys thereof).

In some embodiments, a sensor can include one or more identificationcomponents that are configured to communicate identifying information toanother component of a system (e.g., to a component of an opticalchassis, to a processor, etc.). For example, in some embodiments, asensor can include an identification component that provides an opticalchassis with information regarding, e.g., a type of semitransparent filmdisposed on the sensing surface of the sensor, a configuration of coatedand non-coated regions on a sensing surface of the sensor, aconfiguration of facets in the sensor, etc. In some embodiments, asystem is configured to respond to identifying information communicatedby a sensor. For example, in certain embodiments, a system can beconfigured to receive identifying information from a sensor, and inresponse, configure the system to carry out a particular method ofanalysis (e.g., configure the system to generate one or more opticalsignals having a particular wavelength or wavelengths). Identificationcomponents in accordance with embodiments of the invention can have anysuitable structure, and can include, for example, bar codes, magneticstrips, computer-readable chips, and the like. Systems in accordancewith embodiments of the invention can be configured with a correspondingidentification component that is configured to receive and/or identifyidentification information from an identification component on a sensor.

Aspects of the subject sensors include retention components that areconfigured to retain a sensor in a fixed position with respect toanother component of a subject system (e.g., an optical chassis,described further herein). Retention components in accordance withembodiments of the invention can have any suitable shape and dimensions,and can take the form of, e.g., tabs or flanges that extend from one ormore portions of a subject sensor. In some embodiments, a sensor caninclude a retention component that is configured to removably couple thesensor to another component, such as, e.g., an optical chassis. In someembodiments, a sensor is configured to be removably coupled and/orde-coupled to an optical chassis in a touchless, or aseptic manner,meaning that an operator can accomplish the coupling of the sensor tothe optical chassis without compromising the sterility of the sensor,and can accomplish de-coupling the sensor from the optical chassiswithout having to physically contact the sensor.

Aspects of the subject systems include one or more sensor mountingcomponents that are configured to facilitate aseptic handling of asensor, as well as coupling (e.g., removable coupling) of the sensor toan optical chassis. For example, in certain embodiments a sensormounting component is configured to hold a sensor in an aseptic manner,allow a user to couple the sensor to an optical chassis, and thendisengage from the sensor, leaving the sensor coupled to the opticalchassis in an aseptic manner. Sensor mounting components in accordancewith embodiments of the invention can have any suitable dimensions, andin some embodiments include a surface that is complementary to at leasta portion of a sensor. In some embodiments, a sensor mounting componentis configured to cover at least a portion of an external surface of asensor so that the covered portion of the sensor is not accessible to anexternal environment until the sensor mounting component is disengagedfrom the sensor. In some embodiments, a sensor mounting component isadapted for sterilization via any suitable technique, and is adapted tomaintain its functionality after the sterilization has been completed.Sterilization techniques are well known in the art and include, e.g.,heat sterilization, gamma irradiation, chemical sterilization (e.g.,ethylene oxide gas sterilization), and many others. Aspects of theinvention include sensor mounting components that are adapted forsterilization without altering their functionality in any appreciablemanner. In some embodiments, a sensor mounting component is configuredto allow sterilization of a sensor while the sensor and the sensormounting component are coupled to one another.

Aspects of the subject sensors include one or more kinematic mountingcomponents that are configured to provide a number of constraints thatis equal to the number of degrees of freedom of the component beingmounted. For example, for a three dimensional object having six degreesof freedom, kinematic mounting components that provide six constraintscan be used to mount a sensor on an optical chassis (described furtherbelow).

Aspects of the subject sensors include one or more alignment componentsthat are configured to align the sensor with one or more components ofan optical chassis (described further below). In some embodiments, analignment component can comprise a tapered centering component that isconfigured to align a sensor with an optical chassis.

The subject sensors can be made from any of a variety of suitablematerials, including but not limited to glass, optical grade plastics,polymers, combinations thereof, and the like. Non-limiting examples ofsuitable materials include polymethylmethacrylate (PMMA), polycarbonate(PC), polystyrene (PS), cyclo-olefin polymers (e.g., ZEONEX® E48R),sapphire, diamond, quartz, zircon (zirconium), and the like, or anycombination thereof. In some embodiments, a material that is used tomake a subject sensor can have a refractive index that ranges from about1.2 up to about 2.0, such as 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27,1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39,1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51,1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63,1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75,1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87,1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, or1.99. Those of skill in the art will recognize that any material havingsuitable optical properties can be used in the subject sensors. Sensorsin accordance with embodiments of the invention can be fabricated usingany suitable technique, such as machining, 3D-printing, and/or molding(e.g., injection molding). In some embodiments, a sensor can befabricated using a suitable technique, and can then be further processedto deposit one or more compositions on a surface of the sensor (e.g., asemitransparent film, adhesion layer, or a reflective coating). In someembodiments, a sensor is disposable, and can be discarded after one ormore uses. In some embodiments, a sensor is adapted for repeated use,for example, is adapted to be cleaned and sterilized following use, andthen used again.

As reviewed above, aspects of the invention include sensors that areconfigured to direct a first optical signal to interact with a sensingsurface at a first incident angle, and to direct a second optical signalto interact with the sensing surface at a second incident angle so thatdata from the sensing surface for two different test media (e.g., airand a biological sample, e.g., a tear film) can be captured in the samefield of view, or image frame, of a detection component. In someembodiments, a sensor is configured to direct a first optical signal tointeract with a sensing surface over a narrow range of first incidentangles, and to direct a second optical signal to interact with thesensing surface over a narrow range of second incident angles in orderto generate data in the same field of view, or image frame, of adetection component, as reviewed above. In some embodiments, a narrowrange of incident angles spans a number of degrees ranging from about 2to about 10 degrees, such as about 3, 4, 5, 6, 7, 8 or 9 degrees.

Without being held to theory, a range of first and second incidentangles that are chosen for a sensor depends on the optical properties ofthe material that is used to fabricate the sensor, as well as theexternal medium to be analyzed by the sensor. As such, a first andsecond incident angle, or a first and second narrow range of incidentangles, can differ for sensors that are composed of different materials,and a range of incident angles for a given sensor can be based on theanticipated refractive index of a sample being analyzed (e.g., abiological sample). In some embodiments, a sensor is configured to havea dynamic range of incident angles of clinical significance, wherein thesensor is configured to direct one or more optical signals to interactwith a sensing surface over a range of incident angles that facilitateanalysis of a sample and provide data having clinical significance(e.g., data that facilitate the determination of the osmolarity of abiological sample, e.g., a tear film). Those of skill in the art willappreciate that different first and second incident angles, or rangesthereof, can be selected based on, e.g., the optical properties of thematerial that is used to fabricate the sensor, the properties of theexternal media that will be brought into contact with the sensingsurface (e.g., a biological sample and/or a reference medium), theproperties of the semitransparent film, and/or the properties of theadhesion layer (if present), in order to generate data in the same fieldof view of a detection component from the sensing surface for differenttest media, or different combinations of reference and test media (e.g.,air and water, air and tear fluid, etc.). In some embodiments, a rangeof incident angles broadly spans about 35 degrees to about 75 degrees,such as about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73 or 74 degrees.

In some embodiments, a sensor, when coupled with an optical chassis (asdescribed below) can be formed into a benchtop system that is configuredfor use in a laboratory setting, e.g., in a clinical laboratory setting.In some embodiments, a sensor, when coupled with an optical chassis (asdescribed below) can be formed into a hand-held system. In a preferredembodiment, a hand-held system has dimensions that are similar to thoseof a pen. In use, a hand-held system can be held by, e.g., a physician,and contacted with a sample undergoing analysis.

In some embodiments, a sensor is adapted for sterilization via anysuitable technique, and is adapted to maintain its functionality afterthe sterilization has been completed. Sterilization techniques are wellknown in the art and include, e.g., heat sterilization, gammairradiation, chemical sterilization (e.g., ethylene oxide gassterilization), and many others. Aspects of the invention includesensors that are adapted for sterilization without altering theirfunctionality in any appreciable manner.

Aspects of the invention include kits that contain a plurality ofsensors. In some embodiments, a kit can contain a plurality of identicalsensors. In some embodiments, a kit can contain two or more sensorshaving different characteristics (e.g., a plurality of a first type ofsensor, and a plurality of a second type of sensor). Kits in accordancewith embodiments of the invention can comprise any suitable packaging,for example, can comprise airtight packaging (e.g., hermetically sealedpackaging), vacuum sealed packaging, and the like. In certainembodiments, a kit can be sterile (e.g., the contents of the kit aresterile, and the kit packaging is configured to maintain the sterilityof the contents). In some embodiments, a kit can comprise a plurality ofsensors, wherein each individual sensor is separately sealed in sterilepackaging. In some embodiments, a kit is not sterile, but is adapted forsterilization so that the kit can be sterilized at a point of use, e.g.,at a clinician's office or at a hospital. In some embodiments, a kit canfurther include one or more sensor mounting components, as describedherein.

In some embodiments, a sensor is storage stable and can be stored for anextended period of time, such as one to two years or more, whilemaintaining its functionality. In certain embodiments, a sensor can beprovided in a kit with suitable packaging so that the sensor remainsstorage stable for an extended period of time. For example, in someembodiments, a sensor can be provided in airtight packaging or vacuumsealed packaging to facilitate storage stability for an extended periodof time.

In one preferred embodiment, a sensor is fabricated from a cyclo-olefinpolymer and has a frustoconical, concave shape, having an interiorsurface and an exterior surface, wherein the sensor comprises two facetson the interior surface and four facets on the exterior surface, as wellas a sensing surface located on the exterior surface, and wherein thefacets are configured to direct a first optical signal to interact withthe sensing surface at an incident angle of about 42 degrees, and todirect a second optical signal to interact with the sensing surface atan incident angle of about 64 degrees. In this preferred embodiment,data from both air and water, or from both air and tear fluid, can becollected in the same field of view, or image frame, of a detectioncomponent, thereby providing an internal reference within the image thatcan be used in analysis.

In another preferred embodiment, a sensor is fabricated from acyclo-olefin polymer and has a frustoconical, concave shape, having aninterior surface and an exterior surface, wherein the sensor comprisestwo facets on the interior surface and four facets on the exteriorsurface, as well as a sensing surface located on the exterior surface ofthe sensor, and wherein the facets are configured to direct a firstoptical signal to interact with a sensing surface over a narrow range ofincident angles that ranges from about 40 to about 45 degrees, and isconfigured to direct a second optical signal to interact with thesensing surface over a narrow range of incident angles that ranges fromabout 62 to about 67 degrees.

Turning now to FIG. 22, an illustration of a sensor in accordance withone embodiment of the invention is provided. The depicted embodiment isan injection molded clear plastic sensor with a sensing surface thatcomprises a gold film.

FIG. 23 is an illustration of another sensor in accordance withembodiments of the invention. In the depicted embodiment, the sensorcomprises a sensing surface with a gold film. An upper portion of thedepicted sensor functions as an SPR prism. A middle portion of thedepicted sensor is a skirt portion, and the a lower portion of thedepicted sensor is a base portion that connects to an optical chassis(described further herein).

FIG. 24 is another illustration of a sensor in accordance withembodiments of the invention. In the depicted embodiment, the sensor isconfigured to direct a first optical signal to interact with the sensingsurface at an incident angle of about 42.04 degrees, and is configuredto direct a second optical signal to interact with the sensing surfaceat an incident angle of about 64.44 degrees.

FIG. 25 is another illustration of a sensor in accordance withembodiments of the invention. In the depicted embodiment, the sensor isconfigured to direct a first optical signal to interact with the sensingsurface at an incident angle of about 42.04 degrees, and is configuredto direct a second optical signal to interact with the sensing surfaceat an incident angle of about 64.44 degrees. Further indicated are: agold coating on the sensing surface, an elliptical outer surface of thesensor, an optional curved lower surface of the sensor, a point sourceLED and a beam splitter.

FIG. 42, Panel A is a side view of a sensor in accordance withembodiments of the invention having a frustoconical, concave shape withan internal surface and an external surface. In the depicted embodiment,an outer surface of the sensor has 4 reflecting facets and a taperedcentering component that mates to an optical chassis. Panel B is abottom view of the sensor, showing 2 facets on the internal surface ofthe sensor. Also depicted are retention components and kinematicmounting components.

FIG. 43 is a perspective view of the sensor depicted in FIG. 42. Aplurality of retention fixtures are visible, as well as the sensingsurface and 4 reflecting facets on the external surface of the sensor.

FIG. 44, Panel A is a side view of a sensor in accordance withembodiments of the invention having a frustoconical, concave shape withan internal surface and an external surface. In the depicted embodiment,an outer surface of the sensor has 4 reflecting facets and a taperedcentering component that mates to an optical chassis. Panel B is sideview of a sensor, showing a dashed line that indicates the flow ofmaterial through a mold during the process of fabricating the sensor.Also depicted are kinematic mounting locations.

FIG. 45 is a top, end view of a sensor in accordance with embodiments ofthe invention. The depicted sensor includes a sensing surface thatcomprises coated and non-coated regions. Also depicted are threeretention components, or tabs, that are configured to removably couplethe sensor to an optical chassis.

FIG. 47 is a transparent, perspective view of a sensor in accordancewith embodiments of the invention.

Optical Chassis

As summarized above, aspects of the invention include an optical chassisthat comprises an optical signal generating component and a detectioncomponent. In some embodiments, an optical chassis can comprise anoptical signal manipulation component. Each of these aspects aredescribed in greater detail below.

Aspects of the invention include one or more optical signal generatingcomponents that are configured to generate an optical signal. In someembodiments, an optical signal generating component can include anoptical source that generates an optical signal, such as, e.g., a laser,a light emitting diode (LED), a point-source LED, or a white lightsource with a spectral filter. In some embodiments, an optical chassiscan include a number of optical signal generating components rangingfrom 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9 optical signal generatingcomponents.

Optical signal generating components in accordance with embodiments ofthe invention can be configured to generate light having any suitablewavelength (e.g., may have any suitable emission spectrum), ranging fromUV light, to visible light, to infrared light. In some embodiments, anoptical signal can have a wavelength that ranges from about 300 nm toabout 1,500 nm, such as about 325, 350, 375, 387, 393, 400, 425, 433,445, 450, 467, 475, 488, 490, 492, 494, 495, 500, 502, 505, 510, 516,517, 520, 525, 545, 550, 567, 573, 574, 575, 585, 596, 600, 603, 605,611, 625, 633, 645, 650, 655, 667, 670, 673, 675, 690, 694, 700, 725,750, 775, 800, 825, 850, 855, 875, 900, 925, 940, 950, 975, 1,000,1,025, 1,033, 1,050, 1,060, 1,075, 1,100, 1,125, 1,150, 1,175, 1,200,1,225, 1,250, 1,270, 1,275, 1,300, 1,325, 1,350, 1,375, 1,400, 1,425,1,450, or 1,475 nm. In some embodiments, an optical signal can have awavelength of about 855 nm. In some embodiments, an optical source canhave a wavelength of about 950 nm.

Optical signal generating components in accordance with embodiments ofthe invention can be configured to generate optical signals in a varietyof ways. For example, in some embodiments, an optical signal generatingcomponent is configured to generate an optical signal in a continuousmanner. In some embodiments, one or more optical signal generatingcomponents can be configured to simultaneously generate optical signalshaving two different wavelengths. In some embodiments, an optical signalgenerating component is configured to generate flashing optical signalsthat can be measured in a gated manner. In some embodiments, an opticalsignal generating component is configured to generate an optical signalhaving a single wavelength. In some embodiments, an optical signalgenerating component is configured to generate a plurality of opticalsignals having different wavelengths, such that the same optical signalgenerating component can generate optical signals of two or moredifferent wavelengths.

In some embodiments, an optical chassis comprises an opto-mechanicalreference (OMR) component that is configured or adapted to place aphysical obstruction in the path of one or more optical signals. Thephysical obstruction creates one or more reference signals that can bedetected and analyzed by a detection component. In some embodiments, anOMR is configured to create a vertical or a horizontal obstructionwithin one or more optical signals, such that a vertical or horizontalshadow, or blocked region of the optical signal, can be detected by adetection component. In some embodiments, an OMR is configured to createa combination of vertical and horizontal obstructions within one or moreoptical signals, such that a combination of vertical and horizontalshadows, or blocked regions of the optical signal, can be detected by adetection component. In some embodiments, an opto-mechanical componentis configured to create a circular or elliptical obstruction within oneor more optical signals, such that a circular or elliptical shadow, orblocked region of the optical signal, can be detected by a detectioncomponent. Aspects of the subject methods involve detecting a pixelposition of one or more features of an OMR signal, and using the pixelposition of the one or more features of the OMR signal in a calibration,quality control, and/or data analysis procedure.

Aspects of the invention include a detection component that isconfigured to detect one or more optical signals from the subjectsensors, and to generate data therefrom. In some embodiments, adetection component is configured to detect one or more optical signalsfrom a subject sensor, and to generate an image (e.g., a digital image)of the data for analysis. In some embodiments, a detection component isconfigured to generate a plurality of images from one or more opticalsignals. In some embodiments, a detection component is configured togenerate a plurality of images per second, such as 10, 20, 30, 40, 50,60, 70, 80, 90, or a 100 or more images per second. In some embodiments,a detection component comprises a video recording component (e.g., avideo camera) that is configured to generate a video of one or moreoptical signals that are received from a sensor. In some embodiments, adetection component is configured to capture one or more image frames ofa video, and to subject the one or more image frames to furtherprocessing, as described further below. In certain embodiments, adetection component is configured to begin capturing data before asample is contacted with the sensing surface of a subject sensor, and torapidly capture data immediately following contact of the sample withthe sensing surface. In some embodiments, a detection component isconfigured to begin capturing data concurrently with contact between thesample and the sensing surface, and to rapidly capture data immediatelyfollowing contact of the sample with the sensing surface.

Detection components in accordance with embodiments of the invention areconfigured to receive an optical signal as an input, and to direct theoptical signal to a detector for analysis. In some embodiments, adetection component may be configured to only allow light of a certainwavelength, or of a certain wavelength range, to enter the detectioncomponent. For example, in some embodiments, a detection component caninclude one or more optical filters that are configured to only allowlight of a certain wavelength range to enter the detection component.

In some embodiments, a detection component can include one or moredetectors comprising a photodiode. Photodiodes in accordance withembodiments of the invention are configured to absorb photons of lightand convert the light into electrical current that can be measured. Insome embodiments, a photodiode may include one or more optical filters,lenses, or any other suitable components that may be used to convertlight energy into electrical current for measurement.

In some embodiments, a detection component can include one or morephotomultiplier tubes (PMTs). PMTs in accordance with embodiments of theinvention are configured to detect incident photons by multiplying acurrent produced by an incident light signal.

In some embodiments, a detection component can include one or moreavalanche photodiodes (APDs) or single photon avalanche diodes (SPADs),also known as Gieger-mode avalanche photodiodes, or G-APDs. APDs andSPADs in accordance with embodiments of the invention can detect opticalsignals (such as low intensity signals) down to the single photon levelby exploiting a photon-triggered avalanche current in a semiconductordevice to detect incident electromagnetic radiation.

In some embodiments, a detection component can include one or morestreak cameras that operate by transforming a temporal profile of alight pulse into a spatial profile on a detector by causing atime-varying deflection of the light pulse across the detector.

In some embodiments, a detection component can include one or moredetectors with an image sensor. Image sensors in accordance withembodiments of the invention are configured to convert an optical imageinto an electronic signal. Examples of image sensors include, but arenot limited to, charge coupled devices (CCDs) and complementarymetal-oxide semiconductor (CMOS) or N-type metal-oxide semiconductordevices. In some embodiments, an image sensor can be an active pixelsensor (APS).

In some embodiments, a detection component can include one or morecameras. In some embodiments, a camera is a CCD camera or a scientificCMOS camera (sCMOS) providing extremely low noise, rapid frame rates,wide dynamic range, high quantum efficiency (QE), high resolution, and alarge field of view. Such cameras are commercially available fromscientific technology vendors.

In some embodiments, a detection component can include one or morelinear array sensors (LASs). Linear array sensors in accordance withembodiments of the invention comprise a linear array of integratingphotosensing pixels, which are configured to measure incident light overa defined exposure time, and to generate a voltage or digital outputthat represents the light exposure of each pixel in the array. LASs areknown in the art and are generally available in a variety of dimensionsand pixel resolutions (DPI). In some embodiments, an analog output of anLAS can be directly interfaced to an analog-to-digital converter (ADC)to carry out digital signal processing.

In some embodiments, a detection component is configured to generate animage of one or more optical signals received from a subject sensor, andto convert or render the image into a digital image comprising aplurality of pixels that are organized on a coordinate system in animaging array. In some embodiments, a digital image can have atwo-dimensional coordinate system, e.g., an x,y coordinate systemassociated therewith, wherein each pixel in the digital image isassigned an x,y coordinate. In certain embodiments, a detectioncomponent can generate a gray-scale digital image, wherein each pixel inthe digital image is assigned a gray-scale value corresponding to arange of gray shades from white to black. In some embodiments, adetection component can generate a color digital image, wherein eachpixel in the digital image is assigned a color. In some embodiments, anumber of pixels in the x direction of the imaging array ranges fromabout 500 to about 4,000 or more, such as about 1,000, 1,500, 2,000,2,500, 3,000, or 3,500 or more. In some embodiments, a number of pixelsin they direction of the imaging array ranges from about 500 to about4,000 or more, such as about 1,000, 1,500, 2,000, 2,500, 3,000, or3,500. Any detection component capable of generating an image from oneor more signals that are received from a subject sensor can be used inaccordance with the subject systems and methods.

Aspects of the subject systems include optical signal manipulationcomponents that are configured to manipulate one or more characteristicsof an optical signal. Examples of optical signal manipulation componentsinclude, but are not limited to, mirrors, lenses (e.g., cylindricallenses, doublet lenses, collimating lenses), beam splitters, prisms(e.g., beam translating prisms), diffraction gratings, photomultipliertubes, optical filters (e.g., optical filters that reduce externalambient light, such as, e.g., long pass filters, baffle components, andthe like that can reduce or eliminate ambient light), beam shapingoptics, optical waveguides, polarizers, spatial filters/spatialapertures, and the like. Optical signal manipulation components inaccordance with embodiments of the invention can include any suitablenumber of individual components, including, in some embodiments, aplurality of the same individual component (e.g., a plurality ofphotomultiplier tubes, a plurality of polarizers, etc.).

In some embodiments, aspects of the subject systems include one or morespatial apertures. Spatial apertures (also known as spatial filters) inaccordance with embodiments of the invention are components that areconfigured to remove aberrations in a light beam due to imperfections orvariations in one or more optical components of the system. In someembodiments, a spatial aperture includes an aperture, or opening, thatis placed in the optical path of an optical signal and allows a desiredportion of the optical signal to pass through the aperture, whileblocking light that corresponds to an undesired portion or structure ofthe optical signal. Spatial apertures in accordance with embodiments ofthe invention can include a small circular aperture, or “pinhole”aperture, that allows light to pass through. In some embodiments, aspatial aperture has an aperture whose diameter ranges from 50 μm to 500μm, such as 100, 150, 200, 250, 300, 350, 400 or 450 μm. In certainembodiments, a spatial aperture may include an aperture whose size isvariable, and the subject methods may include varying the size (e.g.,varying the diameter) of the spatial aperture. In certain embodiments, aspatial aperture may include an aperture whose size can be varied from50 μm to 500 such as 100, 150, 200, 250, 300, 350, 400 or 450 μm.

In certain embodiments, an optical signal manipulation component can beused to shape an optical signal from an optical source to create acollimated optical signal. In certain embodiments, one or more opticalcomponents may be used to shape an optical signal into a collimatedoptical signal. For example, in some embodiments, an optical collimatinglens or a collection of lenses may be positioned in the path of anoptical signal and used to shape the optical signal from the opticalsource into a collimated optical signal.

In some embodiments, an optical signal manipulation component caninclude one or more polarizers that are configured to polarize anoptical signal. Polarization can be p-polarization (i.e., transversemagnetic (TM) polarization), or can be s-polarization (i.e., transverseelectric (TE) polarization), or any combination thereof. In someembodiments, an optical signal manipulation component can include anelliptical polarizer and/or a circular polarizer that are configured topolarize an optical signal.

Aspects of the invention include a controller, processor and computerreadable medium that are configured or adapted to control and/or operateone or more components of the subject systems or sensors. In someembodiments, a system includes a controller that is in communicationwith one or more components of the subject systems or sensors, asdescribed herein, and is configured to control aspects of the systemsand/or execute one or more operations or functions of the subjectsystems, e.g., to carry out one or more methods described herein. Insome embodiments, a system includes a processor and a computer-readablemedium, which may include memory media and/or storage media.Applications and/or operating systems embodied as computer-readableinstructions (or “firmware”, i.e., permanent software that is programmedinto a read-only memory) on computer-readable memory can be executed bythe processor to provide some or all of the functionalities describedherein, including, by not limited to, carrying out one or more of themethod steps described herein, acquiring and processing data obtainedfrom the subject sensors and/or systems, and/or applying one or morealgorithms or other manipulations to the data for analysis. In someembodiments, firmware can include instructions for executing one or moreimage capture sequences that capture one or more images of a medium thatis placed in contact with a sensing surface. In some embodiments, asystem can include software that has instructions for executing one ormore algorithms that can be used for processing of one or more images,analyzing of data from one or more images (e.g., to determine anosmolarity of a test sample), or any combination thereof. In someembodiments, a system can be configured to carry out one or more methodsautomatically. For example, in some embodiments, a system can beconfigured to automatically execute one or more image capture sequencesand/or image or data processing algorithms in response to a particularevent, e.g., coupling of a sensor to an optical chassis, receipt of auser input (e.g., receipt of an activation signal from a user), etc.

In some embodiments, a system includes a user interface, such as agraphical user interface (GUI), and/or one or more user input devicesthat are adapted or configured to receive input from a user, and toexecute one or more of the methods as described herein. In someembodiments, a GUI is configured to display data or information to auser.

In some embodiments, a system includes one or more temperature controlelements that are configured to control the temperature of one or moreportions of a sensor, and/or one or more components of an opticalchassis. For example, in some embodiments, a system includes atemperature controller that is configured to maintain a sensor or anoptical chassis within a target temperature range. Temperature controlelements in accordance with embodiments of a system may includeresistive heaters, thermoelectric heaters or coolers, fans, and thelike.

In some embodiments, a system includes one or more environmentalanalysis components that are configured to measure one or morecharacteristics of an external environment. For example, in someembodiments, a system can include a temperature sensor (e.g., athermometer or thermocouple) that can measure a temperature of theenvironment. In some embodiments, a system can include a pressure sensor(e.g., a barometer) that can measure a pressure (e.g., a barometricpressure) of the environment. In some embodiments, a system can includea humidity sensor (e.g., a hygrometer, a humidity sensor) that canmeasure a humidity of the external environment. In some embodiments, asystem can include a light sensor that can measure the amount of lightin an environment is which the sensor is operated. In some embodiments,a system can include an environmental composition sensor that canmeasure the composition of the environment (e.g., the presence and/orconcentration of one or more chemical species) in which the sensor isoperated. In certain aspects, the subject systems are configured toaccount for, or correct for, one or more characteristics of an externalenvironment, or a combination of multiple external environmentcharacteristics, when analyzing a sample. For example, in someembodiments, a processor is configured to correct for, e.g., an externaltemperature when analyzing a sample. In some embodiments, a processor isconfigured to correct for, e.g., a combination of humidity andenvironmental composition when analyzing a sample.

Aspects of the subject systems also include data exchange features, suchas, e.g., USB ports, Ethernet ports, or other data ports that areconfigured to establish a connection that can be used toexchange/transmit data between two or more components of a system.Aspects of the subject systems also include wireless transmissioncomponents, such as WiFi components, that are configured to wirelesslytransmit data between two or more components of a system. For example,in some embodiments, a system can transmit data obtained from a sensorto a database or repository for storage.

Aspects of the subject systems also include one or more computerprocessors, data storage, and/or database components that can be used tostore and/or analyze data that is acquired by the subject systems. Suchcomponents can be physically connected to other components of thesubject systems, such as, e.g., via a USB connection, or can beconfigured to wirelessly communicate with other components of thesubject systems, e.g., via WiFi connection, or via the Internet. In someembodiments, computer processors, data storage and/or databasecomponents of the subject systems may be remotely located, e.g., may belocated at a physical location that is different from the physicallocation of a sensor.

Aspects of the subject systems can also include one or more powercomponents, such as batteries and/or power cables that are configured toprovide electrical power to the subject systems. Power components inaccordance with embodiments of the invention may be modular and may beconfigured to be removably coupled to the subject systems for purposesof providing power thereto, for example, one or more batteries orbattery packs that are configured to be inserted into or otherwisecoupled to the subject systems. In some embodiments, the subject systemsinclude power cables that are configured to establish electrical contactwith standard power outlets. In some embodiments, a system can include abase unit that is configured to re-charge one or more components of thesystem (e.g., an optical chassis, or a component thereof).

In some embodiments, a system can include one or more antisepticizingcomponents that are configured to sanitize one or more components of asystem. For example, in some embodiments, a system can include a UVlight antisepticizing component that is configured to illuminate one ormore portions of a system with UV light. In some embodiments, anantisepticising component can be disposed in a base unit that isconfigured to re-charge one or more components of a system, as describedabove.

In some embodiments, the various features of the subject systems areformed into a single device that includes a housing formed from suitablematerials, such as plastic, metal, glass or ceramic materials, and anycombinations thereof. For example, in some embodiments, a system thatincludes a sensor and an optical chassis, as described herein, is formedfrom a plastic housing, and various additional components of the systemare located within the housing. In some embodiments, a system is formedinto a single bench-top system that can be used to carry out the subjectmethods, as described further below. In some embodiments, a system isformed into a single, hand-held system that can be carried by a user. Incertain embodiments, a hand-held system is wireless. In certainembodiments, a hand-held system includes a rechargeable batterycomponent. In a preferred embodiment, the features of a system areformed into a wireless, rechargeable, pen-sized device that can be usedto carry out the methods described herein.

In one preferred embodiment, an optical chassis includes four pointsource LEDs as optical signal generating components, wherein two of thepoint source LEDs are configured to emit light having a wavelength ofabout 855 nm, and two of the point source LEDs are configured to emitlight having a wavelength of about 950 nm. In one preferred embodiment,an optical chassis includes a CMOS digital image sensor having about2592×1944 active pixels, and that converts incident light into a digitalelectronic signal by determining an intensity value of light at eachpixel and assigning a gray-scale value to each pixel.

Aspects of the invention include one or more signal processingcomponents that are configured to analyze data obtained from a detectioncomponent. For example, in some embodiments, a signal processingcomponent is configured to identify a region of interest (ROI) of animage that is generated by a detection component. In some embodiments, asignal processing component is configured to generate a mathematicalfunction that corresponds to an average pixel intensity along a givencoordinate direction of an image. For example, in some embodiments, asignal processing component is configured to calculate an average of avertical column pixel intensity for each pixel position along the xcoordinate of an image, and to generate a mathematical functionrepresenting the results. Once generated, the mathematical function canbe analyzed to determine, e.g., an x coordinate that corresponds to arelative minimum or relative maximum value of the mathematical function.

In certain embodiments, a signal processing component is configured toapply one or more noise reduction techniques that serve to reduce oreliminate noise from a signal. For example, in some embodiments, asignal processing component is configured to apply a Gaussian bluralgorithm to reduce noise in a signal. In some embodiments, a signalprocessing component is configured to use derivative signal processingto precisely locate a zero crossing value of a derivative signal.

In some embodiments, a signal processing component is configured toacquire and analyze a plurality of data from a sample over a timeinterval that ranges from about 0.001 seconds up to about 90 seconds,such as about 0.002 seconds, about 0.003 seconds, about 0.004 seconds,about 0.005 seconds, about 0.006 seconds, about 0.007 seconds, about0.008 seconds, about 0.009 seconds, about 0.01 seconds, about 0.02seconds, about 0.03 seconds, about 0.04 seconds, about 0.05 seconds,about 0.06 seconds, about 0.07 seconds, about 0.08 seconds, about 0.09seconds, about 0.1 seconds, about 0.2 seconds, about 0.3 seconds, about0.4 seconds, about 0.5 seconds, about 0.6 seconds, about 0.7 seconds,about 0.8 seconds, about 0.9 seconds, about 1 second, about 2 seconds,about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds,about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds,about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds,about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds,about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds,about 75 seconds, about 80 seconds, or about 85 seconds.

Referring now to FIG. 26, an optical chassis and sensor in accordancewith embodiments of the invention are depicted. In this illustration,various light paths originating at an LED and traveling through thesystem are depicted. The depicted embodiment includes 855 nm and 950 nmwavelength LED optical sources and a 5 facet sensor. In addition, thedepicted optical chassis includes a doublet lens, a cylinder lens, abeam turning mirror and a detection component.

FIG. 27 depicts another optical chassis and sensor in accordance withembodiments of the invention. In this illustration, various light pathsoriginating at an LED and traveling through the system are depicted. Thedepicted embodiment includes 855 nm and 950 nm wavelength LED opticalsources and a sensor. In addition, the depicted optical chassis includesa cylinder lens, a doublet lens and a detection component.

FIG. 28, Panel A depicts another optical chassis and sensor inaccordance with embodiments of the invention. In this illustration, twolight paths originating at an LED and traveling through the system aredepicted. The depicted embodiment includes 855 nm and 950 nm wavelengthoptical sources (each optical source comprising a set of two LEDs) and asensor with a plurality of internal and external facets, as well as asensing surface. In addition, the depicted optical chassis includes acylinder lens, a collimating lens and detection component. Panel B showsa top, end view of the sensing surface of the depicted sensor. Thesensing surface comprises a coated region with a gold coating (e.g., agold semitransparent film coating) disposed in a rectangular orientationalong the center line of the sensing surface. On either side of thecoated region, the sensing surface comprises a non-coated region. PanelC shows a close up illustration of the sensor and its internal facets(n=2) (labeled with circled numerals 1 and 7), its external facets (n=4)(labeled with circled numerals 2, 3, 5 and 6), as well as the sensingsurface (labeled with circled numeral 4).

FIG. 32, Panel A depicts another optical chassis and sensor inaccordance with embodiments of the invention. In this illustration,various light paths originating at an LED and traveling through thesystem are depicted. The depicted embodiment includes 855 nm and 950 nmwavelength LED optical sources and a sensor. In addition, the depictedoptical chassis includes a cylinder lens, a doublet lens and a detectioncomponent. Panel B is a close up illustration of the internal facets ofthe sensor (n=2) (labeled with circled numerals 1 and 5), externalfacets of the sensor (n=2) (labeled with circled numerals 2 and 4), anda sensing surface, labeled with circled numeral 3. In the depictedembodiment, facet 2 is uncoated, facet 4 is coated with a reflectivecoating, and the sensing surface 3 is coated with a semitransparentfilm.

FIG. 34 depicts another optical chassis and sensor in accordance withembodiments of the invention. In this illustration, various light pathsoriginating at an LED and traveling through the system are depicted. Thedepicted embodiment includes 855 nm and 950 nm wavelength LED opticalsources and a sensor. In addition, the illustration depicts the positionof a cylinder lens, a collimating lens, an optical wedge, and adetection component (e.g., a XIMEA® imager).

FIG. 35 depicts a side view of an optical chassis and a sensor inaccordance with embodiments of the invention. In this illustration,various light paths originating at an LED and traveling through thesystem are depicted. The depicted embodiment includes 855 nm and 950 nmwavelength LED optical sources and a sensor. In addition, theillustration depicts a cylinder lens, a collimating lens, an opticalwedge, and a detection component (e.g., a XIMEA® imager). In thisdepicted embodiment, the sensor is operably coupled to the opticalchassis.

FIG. 36 depicts a side view of an optical chassis and a sensor inaccordance with embodiments of the invention. In this depictedembodiment, the length of the optical chassis is approximately 2.181inches.

FIG. 37 depicts a side view of an optical chassis and a sensor inaccordance with embodiments of the invention. In this depictedembodiment, the height of the optical chassis is approximately 0.903inches, and the diameter of the sensor is approximately 0.765 inches.

FIG. 38 depicts a side view of the optical chassis and a sensor that aredepicted in FIG. 37. In the depicted embodiment, the optical chassisincludes a collimating lens, a cylinder lens, an optical wedge and adetection component (e.g., a XIMEA® imager).

FIG. 39 depicts a side view of an optical chassis and a sensor inaccordance with embodiments of the invention. In the depictedembodiment, the optical chassis includes a chassis window, two cylinderlenses, a beam splitter, 850 and 940 nm wavelength LEDs, an opticalwedge, and a detection component (e.g., a XIMEA® imager).

FIG. 40 is a perspective illustration of an optical chassis and a sensorin accordance with embodiments of the invention. In the depictedembodiment, the optical chassis includes 850 and 940 nm wavelength LEDs,a sensor cap locking component, a polarizer and barrel, a control board,and a detection component (e.g., a XIMEA® imager assembly).

FIG. 41 is a side view of an optical chassis and a sensor in accordancewith embodiments of the invention. In the depicted embodiment, theoptical chassis includes 850 and 940 nm wavelength LEDs, a polarizer andbarrel, a control board, a detection component (e.g. a XIMEA® imagerassembly) and a case (LacriPen Case) that surrounds the optical chassiscomponents.

FIG. 46 is a top, end view of a sensor that is removably coupled to anoptical chassis. In the depicted embodiment, the sensing surface of thesensor is shown, comprising a coated surface (gold coated area) and anon-coated surface (uncoated prism area). The depicted sensor alsoincludes three retention components, or retention tabs, that areconfigured to removably couple the sensor to the optical chassis. Thedepicted sensor is configured to twist lock with the optical chassis.

FIG. 48 is an illustration of a benchtop system in accordance withembodiments of the invention. In this depicted embodiment, the systemincludes a hemi-cylinder sensor, a gold coated microscope slide, animage sensor, a beam splitter, 950 and 855 nm wavelength LED opticalsources and collimators, and a circuit board. The depicted embodiment isdisposed in a square housing and is configured to be disposed on, e.g.,a laboratory benchtop during use.

FIG. 49 is a perspective view of the benchtop system depicted in FIG.48.

FIG. 50 is a labeled perspective view of the benchtop system depicted inFIGS. 48 and 49. The depicted embodiment shows a hemi-cylinder sensor, agold coated microscope slide, an image sensor, a beam splitter, 950 and855 nm wavelength LEDs, and a circuit board.

FIG. 51 is an image of a housing and an accompanying cover plate thatcan be used to house a benchtop system as described in FIGS. 48-50.

METHODS OF USE

Aspects of the invention include methods of analyzing a sample using thesubject sensors and systems to determine, e.g., the osmolarity of thesample. As depicted in FIG. 1, the average osmolarity of tears in normaleyes differs from the average osmolarity of tears in dry eyes, and assuch, can serve as a diagnostic predictor of dry eye disease. Thesubject methods involve contacting a sensing surface of a sensor with amedium to be tested (e.g., a reference medium, or a test sample havingan unknown osmolarity) for a sufficient period of time to carry out oneor more of the subject methods. In some embodiments, a subject methodcan be carried out in a time period that is about 90 seconds or less,such as 80 seconds, 70 seconds, 60 seconds, 50 seconds, 40 seconds, 30seconds, 20 seconds, 10 seconds, 5 seconds, 4 seconds, 3 second, 2second, or 1 second or less, such as 0.5 seconds, 0.4 seconds, 0.3seconds, 0.2 seconds, or 0.1 seconds or less, such as about 0.09seconds, 0.08 seconds, 0.07 seconds, 0.06 seconds, 0.05 seconds, 0.04seconds, 0.03 seconds, 0.02 seconds, or 0.01 seconds or less, such asabout 0.009 seconds, 0.008 seconds, 0.007 seconds, 0.006 seconds, 0.005seconds, 0.004 seconds, 0.003 seconds, 0.002 seconds, or 0.001 secondsor less.

In some embodiments, the subject methods involve determining theosmolarity of a biological sample obtained from a patient or subject.The information can be used to assist a care giver in diagnosing thepatient or subject with a condition or disorder (e.g., dry eye disease)based on the results of the analysis. For example, in some embodiments,if a tear film of a patient is determined to have an osmolarity orsalinity value in a particular range, then the care giver can diagnosethe patient with dry eye disease.

The subject methods can be used to determine the osmolarity of anysuitable biological sample. Biological samples that can be analyzedusing the subject methods include, without limitation: blood, plasma,serum, sputum, mucus, saliva, urine, feces, gastric and digestive fluid,tears, nasal lavage, semen, vaginal fluid, lymphatic fluid, interstitialfluids derived from tumorous tissue, ascites, cerebrospinal fluid,sweat, breast milk, synovial fluid, peritoneal fluid, and amnioticfluid.

Any suitable volume of sample can be used in conjunction with thesubject methods. In some embodiments, the volume of a sample ranges fromabout 5 nanoliters (nL) up to about 1 milliliter (mL), such as about 25,50, 75, or 100 nL, such as about 200, 300, 400, 500, 600, 700, 800, 900or 1,000 nL, such as about 5, 25, 50, 75 or 100 microliters (μL), suchas about 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 μL. In someembodiments, a sensing surface of a sensor is contacted directly to asample, e.g., is placed in direct contact with the sample. In someembodiments, a sensing surface of a sensor is contacted directly to abiological sample without having to physically separate the sample fromthe patient. For example, in some embodiments, a sensing surface iscontacted directly to a tear fluid of a patient while the tear fluidremains in or on the patient's eye. In some embodiments, a sensingsurface is contacted directly to a patient's blood (e.g., in an openwound) without physically separating the blood from the patient. In someembodiments, a sensing surface is contacted directly to a patient'ssaliva without physically removing the saliva from the patient's mouth.

Aspects of the methods involve contacting a sensing surface of a sensorwith a sample (e.g., a biological sample) and directing an opticalsignal having a first wavelength to interact with the sensing surface ata first incident angle and over a first time interval to generate asignal (e.g., an SPR signal) in response. In some embodiments, themethods involve directing a second optical signal having a secondwavelength to interact with the sensing surface at the first incidentangle over a second time interval while the sensing surface is incontact with a sample. In some embodiments, the first and second timeintervals are the same. In some embodiments, the first and second timeintervals are different. In some embodiments, the first and secondoptical signals are directed to interact with the sensing surfaceconcurrently, whereas in some embodiments, the first and second opticalsignals are directed to interact with the sensing surface in a gatedmanner.

Aspects of the methods further involve generating a series of images ofthe SPR signals over the time intervals, and determining a series ofpixel positions that correspond to a minimum value of the SPR signalsover the time intervals. In some embodiments, the pixel positions thatcorrespond to the minimum value of the SPR signals over the timeintervals are used to generate a mathematical function that plots thepixel position of the minimum value of the SPR signals versus time,referred to herein as an SPR function. In some embodiments, the methodsinvolve comparing the SPR function to the pixel position of at least onereference feature to generate a reference-corrected SPR function. Incertain embodiments, the methods involve comparing one or morecharacteristics of a first SPR function, which is generated from a firstoptical signal having a first wavelength, to one or more characteristicsof a second SPR function, which is generated from a second opticalsignal having a second wavelength. In some embodiments, thecharacteristic of the function is a derivative of the function. In someembodiments, the characteristic of the function is a plateau value ofthe function.

Aspects of the methods involve contacting a sensing surface of a sensorwith a reference medium and directing an optical signal having a firstwavelength to interact with the sensing surface at a second incidentangle to generate a signal (e.g., an SPR signal or a critical anglesignal) in response. In some embodiments, the methods involve directingone or more optical signals having different wavelengths to interactwith the sensing surface at the second incident angle while the sensingsurface is in contact with the reference medium.

Aspects of the methods involve measuring critical angle signals as wellas SPR signals that are generated from a sensing surface while thesensing surface is in contact with a reference medium. In someembodiments, an SPR signal is generated by directing an optical signalto interact with a coated region of a sensing surface. In someembodiments, a critical angle signal is generated by directing anoptical signal to interact with a non-coated region of a sensingsurface. In some embodiments, the methods involve directing first andsecond optical signals having different wavelengths to interact with acoated region of a sensing surface to generate first and second SPRsignals. In some embodiments, the methods involve directing first andsecond optical signals having different wavelengths to interact with anon-coated region of a sensing surface to generate first and secondcritical angle signals.

In some embodiments, the methods involve first contacting a sensingsurface of a sensor with a reference medium (e.g., air) and determiningan SPR delta pixel value and/or a critical angle delta pixel value, asdescribed above, and then contacting the sensing surface with a testsample (e.g., a biological sample), and determining the osmolarity ofthe test sample using one or data analysis procedures as describedherein.

In some embodiments, the methods involve directing an optical signal tointeract with a sensing surface at one or more incident angles. Forexample, in some embodiments, the methods involve directing a firstoptical signal to interact with a sensing surface at a first incidentangle, and directing a second optical signal to interact with a sensingsurface at a second incident angle. In some embodiments, the methodsinvolve directing one or more optical signals to interact with a sensingsurface at a different incident angle, depending on the type of mediumthat is in contact with the sensing surface. For example, in someembodiments, the methods involve contacting a sensing surface with atest sample (e.g., a biological sample) and directing one or moreoptical signals to interact with the sensing surface at a first incidentangle, and contacting the sensing surface with a second medium (e.g., areference medium) and directing one or more optical signals to interactwith the sensing surface at a second incident angle. In someembodiments, the methods involve first contacting the sensing surfacewith a reference medium (e.g., air) to calibrate the sensor, verify oneor more quality parameters of the sensor, or to obtain one or morereference values from the reference medium, and then contacting thesensing surface with a test sample (e.g., a biological sample, e.g., atear fluid) and determining the osmolarity of the test sample.

In certain embodiments, the methods involve directing optical signals ofdifferent wavelengths to interact with a sensing surface. As reviewedabove, the subject systems are configured to generate optical signalshaving any wavelength ranging from about 300 to about 1,500 nm. In someembodiments, the methods involve generating a first optical signalhaving a wavelength of about 855 nm, and generating a second opticalsignal having a wavelength of about 950 nm. In some embodiments, aplurality of optical signals can be directed to interact with a sensingsurface simultaneously. For example, in some embodiments, two or moreoptical signals having different wavelengths are directed to interactwith a sensing surface simultaneously. In some embodiments, a pluralityof optical signals can be directed to interact with a sensing surface ina gated manner.

Aspects of the methods involve measuring changes in the intensity of oneor more optical signals that are reflected from the sensing surface as afunction of time while a test sample (e.g., a biological sample) is incontact with the sensing surface. Without being held to theory, theinventors have determined that as components of the sample (e.g.,proteins within a biological sample) interact with the sensing surface(e.g., adsorb onto the sensing surface), the refractive index close tothe sensing surface changes, altering the angle of the minimum reflectedlight intensity, or SPR angle. The change in the SPR angle, and/or therate of change of the SPR angle, is proportional to the concentrationand molecular weight of the components of the sample. The position ofthe minimum reflected light intensity, or minimum value of the SPRsignal, can therefore be measured as a function of time, and theresulting data can be analyzed to determine one or more characteristicsof the sample, such as the osmolarity of the sample, by comparison to acalibration data set.

Aspects of the methods involve signal processing of one or more signalsthat are received from a sensing surface (e.g., one or more SPR signalsand/or critical angle signals). In some embodiments, a system includessignal processing capabilities that are configured to process a signalprior to analysis. For example, in some embodiments, the methods involveprocessing a signal to reduce noise prior to analysis. In someembodiments, the methods involve applying a Gaussian blur algorithm to asignal to reduce the amount of noise in the signal. In some embodiments,the methods involve applying low pass filtering to a signal to reducethe amount of noise in the signal.

Aspects of the methods involve detecting a signal using a detectioncomponent. In some embodiments, a detection component is configured togenerate one or more images that are based on a signal received from asensing surface. In some embodiments, a detection component isconfigured to generate a plurality of images from one or more signalsthat are received by an imaging component. For example, in someembodiments, a detection component is configured to generate a pluralityof images per second once a sample (e.g., a reference medium or a testmedium) has been placed in contact with a sensing surface of a sensor.In some embodiments, a detection component is configured to generate aplurality of images per second, such as 10, 20, 30, 40, 50, 60, 70, 80,90, or a 100 or more images per second. In some embodiments, a detectioncomponent is configured to generate a video of one or more opticalsignals that are received from a sensor. In some embodiments, adetection component is configured to capture one or more image frames ofa video, and to subject the one or more image frames to furtherprocessing, as described further below.

In some embodiments, a detection component has a field of view, and animage can be generated from a region of interest (ROI) within the fieldof view. In certain embodiments, the methods involve capturing data froma plurality of signals from a sensing surface in a single image frame.Capturing data from a plurality of signals in a single image frameprovides an internal reference that can be used in the analysis of asample.

Aspects of the methods involve data processing of an image that isgenerated from a detection component. In some embodiments, dataprocessing involves applying a coordinate system (e.g., an x,ycoordinate system) to an image. In some embodiments, each pixel, or aportion thereof, within a generated image can be assigned a specific x,ycoordinate value. In some embodiments, each pixel within an image can beassigned a numerical value related to the intensity or color of light inthe pixel. For example, in some embodiments, each pixel in an image isassigned a gray-scale value. In some embodiments, each pixel in an imageis assigned a color value. In some embodiments, data processing involvesperforming a mathematical operation on a plurality of pixels. Forexample, in some embodiments, data processing involves calculating anaverage gray-scale value of a plurality of pixels. In some embodiments,data processing involves calculating an average gray-scale value of acolumn of pixels at a particular x coordinate on an image.

Aspects of the methods involve generating mathematical functions basedon the data that is captured in an image using a detection component.For example, in some embodiments, the data from an image can beprocessed and transformed into a function that can be analyzed andmanipulated mathematically using standard techniques. In someembodiments, an image is analyzed by determining the average gray-scalevalue of a column of pixels at each x coordinate, and the resulting datais converted into a function, or curve, that mathematically represents asignal from which the data was obtained. Once generated, the functioncan be analyzed or manipulated mathematically to determine itscharacteristics. In some embodiments, a plurality of pixel positions areplotted as a function of time to generate a time-based functionrepresenting, e.g., a change in the minimum value of an SPR signal as afunction of time.

In some embodiments, a function can be analyzed to determine a minimumvalue or a maximum value using standard techniques. For example, in someembodiments, a first and/or second derivative of a function can bedetermined and used to calculate a relative minimum or relative maximumof the function. In some embodiments, a function can be smoothed usingstandard techniques, thereby reducing or diminishing noise in the data.

Aspects of the methods involve analyzing a function that is derived froman SPR signal in order to identify a pixel position corresponding to aminimum value of the function. The minimum value of the functioncorresponds to a reflectivity minimum of an SPR signal, and can be usedin analyzing a sample (e.g., determining the osmolarity of a sample).

Aspects of the methods involve analyzing a function that is derived froma critical angle signal in order to identify a pixel positioncorresponding to a maximum value of the function. The pixel positioncorresponding to the maximum value of the function can be used todetermine the critical angle of the sensor.

In some embodiments, aspects of the methods involve analyzing data thatis obtained from a reference feature. In some embodiments, the referencefeature is an opto-mechanical reference (OMR) feature, and the data thatis obtained from the OMR is one or more pixel positions from a referencesignal that is generated by the OMR. For example, in some embodiments,an OMR creates a reference signal that can be analyzed to determine oneor more parameters of a sample. In certain embodiments, a referencesignal created by an OMR can be used as a fixed reference signal againstwhich changes in an SPR minimum value (e.g., the number of pixels bywhich the SPR minimum value is moved, or shifted) can be measured when asensing surface of a sensor is contacted with a sample, or is contactedwith a plurality of different samples (e.g., an air sample and a watersample, an air sample and a tear fluid sample, etc.). In certainembodiments, a reference signal created by an OMR can be used as a fixedreference signal that can be compared across different sample types(e.g., air and water, air and tear film, water and tear fluid, etc.). Insome embodiments, a reference feature is a data value obtained from oneor more SPR signals, or one or more critical angle signals. For example,in some embodiments, a sensing surface of a sensor is contacted with areference medium, and one or more SPR signals are generated. A pixelposition corresponding to a minimum value of the one or more SPRsignals, or a comparison of such minimum values, can be used as areference feature. In some embodiments, one or more critical anglesignals are generated from a sensor, and a pixel position correspondingto a maximum value of the one or more critical angle signals, or acomparison of such maximum values, can be used as a reference feature.

Aspects of the methods involve comparing pixel positions correspondingto various features of the above-described mathematical functions. Forexample, in some embodiments, a method involves comparing a pixelposition of a minimum value of a function derived from a first SPRsignal to the pixel position of a minimum value of a function derivedfrom a second SPR signal to determine an SPR delta pixel value. The SPRdelta pixel value represents the distance between the minimum values ofthe first and second SPR signals. In some embodiments, the methodsinvolve comparing a pixel position of a maximum value of a functionderived from a first critical angle signal to the pixel position of amaximum value of a function derived from a second critical angle signalto determine a critical angle delta pixel value. The critical angledelta pixel value represents the distance between the maximum values ofthe first and second critical angle signals.

In some embodiments, the methods involve mathematically manipulating adelta pixel value to account for one or more external conditions thatcan impact the operation of a subject sensor. For example, in someembodiments, the methods involve multiplying or dividing a delta pixelvalue by a correction factor in order to account for an externalcondition. As reviewed above, in some embodiments, a subject system caninclude an environmental analysis component that can be used to measureone or more characteristics of the environment in which the sensor isoperating.

In some embodiments, the methods involve verifying a quality parameterof a sensor. For example, in some embodiments, one or morecharacteristics of a signal that is generated by a sensor is evaluatedto determine whether the sensor is of sufficient quality for use. Insome embodiments, one or more characteristics of an SPR signal isevaluated to determine whether the sensor is of sufficient quality foruse. In certain embodiments, a contrast value, shape, or dimension(e.g., height, width, or depth) of an SPR signal (or a data set orfunction derived therefrom) is evaluated to determine if the sensor isof sufficient quality for use. In some embodiments, one or morecharacteristics of a critical angle signal is evaluated to determinewhether the sensor is of sufficient quality for use. In certainembodiments, a contrast value, shape, or dimension (e.g., height, width,or depth) of a critical angle signal (or a data set or function derivedtherefrom) is evaluated to determine if the sensor is of sufficientquality for use. In some embodiments, the methods can be used to verifywhether a sensor has, e.g., a sufficient thickness of a semitransparentfilm and/or adhesion layer on the sensing surface, or a sufficientpurity of a material in the semitransparent film and/or adhesion layer.

Aspects of the methods involve comparing one or more data values (e.g.,one or more delta pixel values, one or more corrected delta pixelvalues) to a calibration data set in order to determine a characteristicof a sample (e.g., an osmolarity of a sample). In some embodiments, asystem can include a plurality of calibration data sets that can be usedfor different purposes. In some embodiments, a system includes acalibration data set that includes osmolarity values as a function ofdelta pixel values, and the methods involve comparing a delta pixelvalue to the calibration data set to determine the osmolarity of asample. In some embodiments, a system includes a calibration data setthat includes quality parameter values, and the methods involvecomparing one or more characteristics of a signal that is generated by asensor to the calibration data set to determine whether the sensor is ofsufficient quality for use. In some embodiments, a system includes acalibration data set that includes correction factors for variousexternal environment parameters, and the methods involve comparing ameasured external environment parameter to the calibration data set todetermine an appropriate correction factor, and then mathematicallymanipulating a delta pixel value to apply the correction factor.

In some embodiments, a method involves operably connecting a sensor toan optical chassis. In certain embodiments, a method involves removablycoupling a sensor to an optical chassis, carrying out an analysismethod, as described herein, and then removing the sensor from theoptical chassis. In some embodiments, the methods involve asepticallycoupling a sensor to an optical chassis. In some embodiments, themethods involve aseptically de-coupling a sensor from an opticalchassis.

Aspects of the methods involve the analysis of any suitable sample. Insome embodiments, a sample is a gaseous or a liquid medium. In certainembodiments, a medium can be a calibration medium, having a knownosmolarity value. For example, in some embodiments, the methods involvecontacting a sensor with a medium having a known osmolarity, directingone or more optical signals to interact with the sensing surface, anddetecting one or more signals resulting therefrom (e.g., detecting anSPR signal or a critical angle signal). In some embodiments, a samplecan be a reference medium (e.g., a medium against which a test medium orsample will be compared). In some embodiments, a reference medium can beair (e.g., the air in a room where the sensor is used). In someembodiments, a sample is a liquid medium, e.g., water. In someembodiments, a sample can be a biological sample, as described above. Insome embodiments, the methods involve contacting a sensing surface of asensor with a sample, and maintaining contact between the sample and thesensing surface while at least some of the method steps are carried out.

In a preferred embodiment, a method involves contacting a sensingsurface of a sensor with a tear fluid from a subject. A first opticalsignal having a wavelength of about 855 nm is directed to interact withthe sensing surface at an incident angle of about 64 degrees to generatea first SPR signal over a first time interval. The first SPR signal isdetected over the first time interval with the detection component. Aplurality of images of the signal are recorded over the first timeinterval, and the pixel position corresponding to the minimum value ofthe SPR signal is plotted as a function of time to generate a firsttime-based SPR function.

Next, a second optical signal having a wavelength of about 950 nm isdirected to interact with the sensing surface at the same incident angleof about 64 degrees to generate a second SPR signal over a second timeinterval. The second SPR signal is detected over the second timeinterval with the detection component. A plurality of images of thesignal are recorded over the second time interval, and the pixelposition corresponding to the minimum value of the second SPR signal isplotted as a function of time to generate a second time-based SPRfunction.

Next, the first and second time-based SPR functions are compared to atleast one reference feature to a generate a first and a secondreference-corrected SPR function. One or more characteristics of thefirst and second reference-corrected SPR functions are then analyzed todetermine the osmolarity of the tear fluid. In some embodiments, thereference feature comprises a pixel position of one or more OMRfeatures.

In one preferred embodiment, the methods further comprise contacting thesensing surface of a sensor with air as a reference medium and directinga first optical signal having a wavelength of about 855 nm to interactwith the sensing surface at an incident angle of about 42 degrees togenerate a third SPR signal. The third SPR signal is detected with adetection component that generates an image from the signal. The imageof the signal is processed to generate a mathematical function thatrepresents the third SPR signal, and which does not substantially varywith respect to time. The pixel position corresponding to the minimumvalue of the function is determined.

Next, a second optical signal having a wavelength of about 950 nm isdirected to interact with the sensing surface at the same incident angleof about 42 degrees to generate a fourth SPR signal. The fourth SPRsignal is detected with a detection component that generates an imagefrom the signal. The image of the signal is processed to generate amathematical function that represents the fourth SPR signal, and whichdoes not substantially vary with respect to time. The pixel positioncorresponding to the minimum value of the function is determined.

The pixel positions corresponding to the minimum values of the third andfourth SPR signals are then compared to determine an SPR delta pixelvalue. In some embodiments, the pixel position corresponding to theminimum value of the third or fourth SPR signal is used as a referencefeature that the first and a second reference-corrected SPR functionsare compared to. In some embodiments, the SPR delta pixel value is usedas a reference feature that the first and a second reference-correctedSPR functions are compared to. In some embodiments, a combination of thepixel positions of the minimum values of the third and/or fourth SPRsignals, and/or the SPR delta pixel value, is used as a referencefeature that the first and a second reference-corrected SPR functionsare compared to.

In one preferred embodiment, the methods further comprise contacting thesensing surface of a sensor with air as a reference medium and directinga first optical signal having a wavelength of about 855 nm to interactwith the sensing surface at an incident angle of about 42 degrees togenerate a first critical angle signal. The first critical angle signalis detected with a detection component that generates an image from thesignal. The image of the signal is processed to generate a mathematicalfunction that represents the first critical signal, and which does notsubstantially vary with respect to time. The pixel positioncorresponding to the maximum value of the function is determined.

Next, a second optical signal having a wavelength of about 950 nm isdirected to interact with the sensing surface at the same incident angleof about 42 degrees to generate a second critical angle signal. Thesecond critical angle signal is detected with a detection component thatgenerates an image from the signal. The image of the signal is processedto generate a mathematical function that represents the second criticalangle signal, and which does not substantially vary with respect totime. The pixel position corresponding to the maximum value of thefunction is determined.

The pixel positions corresponding to the maximum values of the first andsecond critical angle signals are then compared to determine a criticalangle delta pixel value. In some embodiments, the pixel positioncorresponding to the maximum value of the first or second critical anglesignal is used as a reference feature that the first and a secondreference-corrected SPR functions are compared to. In some embodiments,the critical angle delta pixel value is used as a reference feature thatthe first and a second reference-corrected SPR functions are comparedto. In some embodiments, a combination of the pixel positions of themaximum values of the first and/or second critical angle signals, and/orthe critical angle delta pixel value, is used as a reference featurethat the first and a second reference-corrected SPR functions arecompared to.

Turning now to FIG. 52, Panel A is an image of an SPR signal acquiredwith air as the reference medium in contact with the sensing surface ofthe sensor. Panel B is a graph of grey value as a function of pixelposition for the optical signal shown in Panel A. Panel C provides twoimages of an SPR signal acquired at two different times, t=0 and t=600seconds. Panel D is a graph showing the pixel position of the minimumvalue of the SPR signal shown in Panel C as a function of time after thesensing surface was contacted with a biological sample (e.g., a tearfluid). Panel E is a graph showing a close-up view of the pixel positionof the minimum value of the SPR signal shown in Panel D over a timeinterval of 60 seconds and obtained using an optical signal having awavelength of 855 nm, and corrected by subtracting the pixel position att=0 seconds from the pixel position of the minimum value of the SPRsignal measured at each indicated time point.

FIG. 53 shows a plurality of data from air and from a sample of tearfluid having an osmolarity of 300 mOsm/L. Panel A, top, shows an imageof an SPR signal acquired with air as the reference medium in contactwith the sensing surface. Panel A, bottom, shows an image of an SRsignal acquired with the tear fluid in contact with the sensing surfacefor 17 seconds. The shift to the right of the the vertical black line,which represents the minimum value of the SPR signal, can be seen. PanelB is a graph showing the change in delta pixel value for the sample ofas a function of time following contact of the sensing surface with thetear fluid. The delta pixel value changed rapidly, following contact ofthe sensing surface with the tear solution. Panel C is a graph showinggrey value as a function of pixel position for the air SPR signal. Thepixel position corresponding to the minimum value of the SPR signal iscircled. Panel D is a graph showing grey value as a function of pixelposition for the tear fluid SPR signal taken 17 seconds after the tearfluid was contacted with the sensing surface. The change in delta pixelvalue between the circle positions in Panel C and Panel D isapproximately 571 pixels. This change in pixel position can be used as adata point in a calibration data set for determining the osmolarity oftear fluid.

FIG. 54 is a graph showing delta pixel value as a function of time fortwo different SPR signals that were obtained from a sample of tear fluidhaving an osmolarity of 300 mOsm/L. The first optical signals has awavelength of 850 nm and second optical signal has a wavelength of 950nm. The results demonstrate a significant different in the delta pixelvalue obtained from the two different optical signals.

FIG. 55 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is80% of the protein content of normal tears, using an optical signalhaving a wavelength of 855 nm. Panel A, top, shows an image of an SPRsignal obtained from water at time zero. The vertical black linerepresenting the SPR minimum value can be seen. Panel A, bottom, shows agraph of grey value as a function of pixel position for the image shownin Panel A, top. The SPR minimum occurs at a pixel position of 869.

Panel B, top, shows an image of an SPR signal obtained from the tearfluid at 15 seconds after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel B, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel B, top. The SPR minimumoccurs at a pixel position of 1041.

Panel C, top, shows an image of an SPR signal obtained from the tearfluid at 10 minutes after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel C, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel C, top. The SPR minimumoccurs at a pixel position of 1043.

Below the Panels, the change in pixel position of 172 pixels betweenwater and the tear fluid after 15 seconds is shown, and the change inpixel position of 174 pixels between water and the tear fluid after 10minutes is shown. The SPR signal from the tear fluid reached a plateauvalue, and the pixel position corresponding to the minimum value of theSPR signal did not substantially change between 15 seconds and 10minutes.

FIG. 56 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is80% of the protein content of normal tears, using an optical signalhaving a wavelength of 950 nm. Panel A, top, shows an image of an SPRsignal obtained from water at time zero. The vertical black linerepresenting the SPR minimum value can be seen. Panel A, bottom, shows agraph of grey value as a function of pixel position for the image shownin Panel A, top. The SPR minimum occurs at a pixel position of 1273.

Panel B, top, shows an image of an SPR signal obtained from the tearfluid at 25 seconds after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel B, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel B, top. The SPR minimumoccurs at a pixel position of 1509.

Panel C, top, shows an image of an SPR signal obtained from the tearfluid at 10 minutes after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel C, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel C, top. The SPR minimumoccurs at a pixel position of 1509.

Below the Panels, the change in pixel position of 236 pixels betweenwater and the tear fluid after 25 seconds and 10 minutes is shown. TheSPR signal from the tear fluid reached a plateau value, and the pixelposition corresponding to the minimum value of the SPR signal did notsubstantially change between 25 seconds and 10 minutes.

FIG. 57 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is120% of the protein content of normal tears, using an optical signalhaving a wavelength of 855 nm. Panel A, top, shows an image of an SPRsignal obtained from water at time zero. The vertical black linerepresenting the SPR minimum value can be seen. Panel A, bottom, shows agraph of grey value as a function of pixel position for the image shownin Panel A, top. The SPR minimum occurs at a pixel position of 994.

Panel B, top, shows an image of an SPR signal obtained from the tearfluid at 15 seconds after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel B, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel B, top. The SPR minimumoccurs at a pixel position of 1132.

Panel C, top, shows an image of an SPR signal obtained from the tearfluid at 10 minutes after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel C, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel C, top. The SPR minimumoccurs at a pixel position of 1166.

Below the Panels, the change in pixel position of 138 pixels betweenwater and the tear fluid after 15 seconds is shown, and the change inpixel position of 172 pixels between water and the tear fluid after 10minutes is shown.

FIG. 58 is a collection of images and graphs showing data obtained fromwater and from a sample of tear fluid having a protein content that is120% of the protein content of normal tears, using an optical signalhaving a wavelength of 950 nm. Panel A, top, shows an image of an SPRsignal obtained from water at time zero. The vertical black linerepresenting the SPR minimum value can be seen. Panel A, bottom, shows agraph of grey value as a function of pixel position for the image shownin Panel A, top. The SPR minimum occurs at a pixel position of 1324.

Panel B, top, shows an image of an SPR signal obtained from the tearfluid at 26 seconds after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel B, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel B, top. The SPR minimumoccurs at a pixel position of 1533.

Panel C, top, shows an image of an SPR signal obtained from the tearfluid at 10 minutes after the tear fluid is contacted with the sensingsurface. The vertical black line representing the SPR minimum value canbe seen. Panel C, bottom, shows a graph of grey value as a function ofpixel position for the image shown in Panel C, top. The SPR minimumoccurs at a pixel position of 1562.

Below the Panels, the change in pixel position of 209 pixels betweenwater and the tear fluid after 26 seconds is shown, and the change inpixel position of 238 pixels between water and the tear fluid after 10minutes is shown.

FIG. 59 is a comparative analysis of a sample of tear fluid having aprotein content that is 80% of the protein content of normal tears, anda sample of tear fluid having a protein content that is 120% of theprotein content of normal tears. Panel A shows the sample having 80% ofthe protein content of normal tears, which was analyzed using an opticalsignal having wavelength A (855 nm) and wavelength B (950 nm). The bargraph in Panel A shows the change in delta pixel value for the 80%protein sample at the first wavelength between 15 seconds and 600seconds, and at the second wavelength between 25 seconds and 600seconds. The data show that for the 80% protein tear fluid, there wasvery little change in the delta pixel value as a function of time foreither wavelength, and that the second wavelength created a greaterresponse in delta pixel value as compared to the first wavelength.

Panel B shows the sample having 120% of the protein content of normaltears, which was analyzed using an optical signal having wavelength A(855 nm) and wavelength B (950 nm). The bar graph in Panel B shows thechange in delta pixel value for the 120% protein sample at the firstwavelength between 15 seconds and 600 seconds, and at the secondwavelength between 25 seconds and 600 seconds. The data show that forthe 120% protein tear fluid, there was a greater change in the deltapixel value as a function of time for both wavelengths, and that thesecond wavelength created a greater response in delta pixel value ascompared to the first wavelength.

FIG. 60 is an analysis of a tear fluid having 100% of the protein innormal tears (i.e., a normal tear fluid sample), analyzed at twodifferent wavelengths (855 nm and 950 nm), and over shorter and longertime intervals. Panel A is a bar graph showing delta pixel value for thesame sample when analyzed using wavelength A (855 nm) for 20 seconds andwavelength B (950 nm) for 17 seconds. At these shorter time intervals,wavelengths A and B provide different delta pixel values, which areclearly distinguished from one another. Panel B is a bar graph showingdelta pixel value for the same sample when analyzed using wavelength A(855 nm) for 20 seconds and wavelength A (855 nm) for 600 seconds. Thechange in delta pixel value as a function of time at the same wavelengthcan clearly be seen.

FIG. 61 is a graph showing the delta pixel value of a salt solution as afunction of osmolarity. The lower series (diamonds) was obtained usingwavelength A (855 nm) and the upper series (squares) was obtained usingwavelength B (950 nm). At an x axis position corresponding to 320mOsm/L, the difference in the y values of the two series is 20 pixels,as shown. At an x axis position corresponding to 340 mOsm/L, thedifference in the y values of the two series is 18 pixels, as shown.This data demonstrates that different changes in delta pixel value areobserved for samples having different osmolarities as a function of thewavelength that is used to conduct the analysis.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES Example 1: Reduction of Optical Noise in a Sensor Using a PointSource LED

Optical noise reduction was achieved in a system by using a point sourceLED as an optical signal generating component. FIG. 3 illustratesclearly that a 638 nm laser diode has substantially higher optical noisethan a red LED (632 nm nominal wavelength), as depicted graphically inthe each of the charts to the right of their corresponding SPR images.Use of a point source LED instead of a laser diode thus reduced theoptical noise in the system.

Example 2: Optimizing Resolution of SPR Signal Measurement

As shown in FIG. 4, longer wavelength optical signals produce narrowerSPR line widths. FIG. 5 illustrates the narrowing of the SPR line withincreasing wavelength as verified experimentally using a simple SPRset-up on an optical table. The decrease of the SPR line width withincreasing wavelength is readily apparent to the naked eye.

It was unclear whether the narrower SPR line width at longer wavelengthswould provide higher SPR resolution in the subject systems, since theangular shift of the SPR minima decreases with longer wavelengths.Consequently, calculations of the angular shift Δθ of the SPR minima fora change in index of refraction by 0.001 refractive index units (“RIU”)and the full width at half maximum (“FWHM”) of the SPR line wereperformed using an online SPR calculator provided by the Research Groupof Prof. Robert M. Corn at the Dept. of Chemistry, University ofCalifornia, Irvine(http://unicorn.ps/uci.edu/calculations/fresnel/fcform.html). The ratioof these two quantities (i.e. AO/FWHM) was defined as the SPRresolution. The result of the calculations was that the improvement ofresolution at a wavelength of 950 nm as compared to 635 nm was in therange of 4 to 5 times. These calculations also showed that there was anegligible difference in resolution obtained using either high indexglass (SF10, n˜1.72) or a lower index glass (BK7, n˜1.52) (see FIG. 6).

Prior to these calculations, the popular scientific folklore was thathigh index prisms provided substantially better SPR performance thanlower index prisms. As a consequence, this well-established scientificfolklore taught away from using injection molded optical plastics asdisposable SPR prisms, since optical plastics generally have relativelylow refractive indices. Thus, based on the above calculations, injectionmolded optical plastics can be used as disposable SPR prisms in thesubject sensors and systems, thereby reducing cost of goods.

Example 3: Derivative Signal Processing

Measurement of tear osmolarity to 1.0 mOsm/L corresponds todetermination of the index of refraction of the tear solution to about 1part in 10⁻⁵ RIU. A common engineering rule of thumb is that theprecision of a measurement should exceed the targeted precision by abouta factor of 10. Consequently it is desirable in a tear osmolaritymeasurement device to have an ultimate index of refraction precision ofabout 1 part in 10⁻⁶ RIU.

Various techniques for determining the location of a SPR line minimumare known in the art. One technique is to fit straight lines to thefalling and rising edges of an SPR line, and is depicted in FIG. 7. Abrief description of the technique is found in U.S. Pat. No. 7,395,103,the disclosure of which is herein incorporated by reference in itsentirety. Another technique, described as a centroid method, is alsodisclosed in U.S. Pat. No. 7,395,103.

It is well known that the points at which the derivative of a functionis zero represent either local maxima or minima of the function.Scientific folklore dismisses use of derivatives to find either themaxima or minima of real world data, since any real world data containsnoise. The commonly held belief is that taking the derivative of noisydata will result in unacceptable noise in the derivative data, thusprecluding accurate determination location of the derivative zerocrossings.

In practice there are three effects that can counteract the effects ofnoise of derivative signal processing for finding the exact location ofthe minima of an SPR curve. The first is to begin with a very low noiseSPR line image. Here, this was accomplished by careful optical design,and by using LEDs rather than lasers for the optical source. Second,moving from visible light sources to near infrared light sources resultsin considerably narrower SPR lines for which the rate of change ofintensity near the SPR minima is rapid, resulting in large derivativesignals relative to any noise in the signal. Finally, any residual noisein the image of the SPR line can be minimized by suitable low passfiltering. Here, a Gaussian blur algorithm was used to diminish anyresidual image noise to acceptable levels.

FIG. 8 presents a typical SPR line image obtained using an 855 nm pointsource LED as the light source. This image was acquired using a 640×480video imager. The image was imported into the application ImageJ imageprocessing software developed at the U.S. National Institutes of Health.Next, 25 pixels of Gaussian blur was applied to the image and anappropriate region of interest was defined for the image, as denoted bythe rectangle in FIG. 8. Within this region of interest (“ROI”) a plotprofile was generated corresponding to the average of the verticalcolumn pixel intensity in the image along the X direction. The result ofthese operations is shown in FIG. 9, which is an ImageJ Plot Profile ofthe region of interest. Finally, data from the plot profile curve can bedifferentiated numerically using well known mathematical techniques inorder to find the positive going zero crossing of the derivative whichprecisely defines the location of the minima of the SPR line (as shownin FIG. 10). Note that the derivative curve shown in FIG. 10 is actualdata derived from the SPR image in FIG. 8. The derivative curve isextremely smooth and exhibits no obvious noise artifacts.

In practice, due to the low level of noise in in the derivative data,the zero crossing of the SPR line's derivative can be located to withina fraction of a pixel using interpolation techniques. FIG. 11illustrates the relative value of the derivative of the SPR image inFIG. 8. Note that there is very little noise in the derivative and thatover the limited range from 220 pixels to 230 pixels the derivative isnearly linear. The zero crossing of the derivative occurs between pixel224 and 225, with coordinates of (224, −0.2943) at pixel 224 and (225,0.1922) at pixel 225. From these values, the exact coordinate of thezero crossing can be determined by linear interpolation, as shown in thegeometry illustrated in FIG. 12. For this example, the zero crossingoccurs precisely at the coordinate (244.6049, 0.0).

FIG. 13 presents the location of the SPR minima for 10 SPR imagessequentially acquired at approximately 1.0 second intervals. There wereno changes in the SPR set-up or other test conditions between each imageacquisition other than time, so that variations in the location of theSPR minima are largely due to the random optical and electronic noisepresent in each acquired image. The images represented by these datawere acquired using an Aptina MT9P031 five megapixel grayscale imagesensor comprised of 2592 horizontal×1944 vertical 2.2 μm square pixels.A separate calibration step, illustrated in FIG. 14, entailed measuringthe SPR line minima pixel locations for ethanol and deionized watercorresponding to a separation of approximately 910 pixels. The index ofrefraction differential between these two liquids is Δn=1.35713(ethanol)−1.3288 (DI water)=0.02833. The result that the Δn per pixel is3.113×10⁻⁵ RIU (FIG. 14). The raw SPR images for the ethanol anddeionized water SPR lines used in this calibration are shown in FIG. 15and FIG. 16, respectively.

Returning to FIG. 13, the overall range of the zero crossing points overthe 10 samples is 0.2662 pixel or ±0.1331 pixel total range about themean pixel value. This corresponds to an overall uncertainty of theindex of refraction of ±4.143×10⁻⁶R.

FIG. 17 depicts SPR osmolarity data acquired and analyzed using thederivative signal processing described above. A series of five precisioncalibrated saline solutions were measured with a miniature opticalbreadboard SPR instrument comprised of a gold coated high index glassSPR prism, an 855 nm point source LED and the Aptina MT9P031 fivemegapixel image sensor. The data captured with this breadboard andprocessed using the derivative signal processing techniquedemonstrated±1.0 mOsm/L precision over an osmolarity range from 295mOsm/L to 348.5 mOsm/L. The saline solutions were independentlycalibrated using the freezing point depression osmolarity measurementtechnique that also has a stated precision of ±1.0 mOsm/L. Clearly theagreement between the freezing point depression method and the SPRtechnique is within the limits of experimental error.

An alternative approach to using low pass filtering (e.g., Gaussianblur) for noise reduction in the derivative signal processing is to usecurve fitting in the region of the SPR minimum to average out noise inthe SPR image. FIG. 18 is an SPR line used to demonstrate this approachto derivative signal processing. It should be noted that the SPR lineprofile in FIG. 18 is distinctly non-symmetric, with the slope on theleft hand side of the SPR minima being substantially less (and oppositein sign) than the slope on the right hand side of the minima. While itis tempting to think of fitting the minima of the SPR line minima to aparabola, in practice this results in a poor fit and low R² value. Theconsequence is that location of the zero crossing found in this manneris displaced from the actual location of the SPR minima. A more accurateapproach is to fit a cubic to the SPR line in the vicinity of its minimaas illustrated in FIG. 19. Generally this results in a R² value nearunity. The resulting cubic equation can then be differentiated, set tozero and solved using the quadratic equation to find the location of theSPR minimum, as further described in FIG. 20.

Example 4: Self-Calibration Sensor Theory

SPR-based analysis can provide extremely precise measurements of thechange of refractive index of a medium (e.g., a gas or a liquid) incontact with the exterior gold surface of the SPR prism. With suitablecare, changes of index of refraction in the range of 1 part in 10⁻⁶ RIUcan be obtained under carefully controlled laboratory conditions (seeFIG. 13). The premise of using SPR for tear osmolarity measurements isthat tear osmolarity and tear index of refraction are linearly related.Saline osmolarity is quite linear with respect to the angular movementof a SPR line, with the linearity shown to be in range off 5.0 mOsm/L to±1 mOsm/L and measurement precision in the range of ±4 10⁻⁶ RIU. Thedata illustrating±1.0 mOsm/L linearity for several precision salinesolutions is shown in FIG. 17.

It should be noted that precisely fitting a line to a series ofprecision calibrated saline solutions is a much easier problem than isthe problem of accurately and precisely determining the salinity (i.e.the index of refraction) of an unknown saline solution. The first casesimply requires determination of the slope of the calibration curve. Thesecond case requires determining both the slope and the y-interceptpoint. Without the aid of external reference solutions, this second caseis extremely difficult to accomplish. External reference solutions arenot practical, since contamination of the gold sensing surface of theSPR instrument is extremely likely to occur.

FIG. 13 provides data from which the RIU per pixel can becalculated—Δn=1.35713−1.3288=0.02853 RIU corresponds to 910 pixels, orΔn/pixel=3.113×10−6 RIU/pixel. The slope of the osmolarity v. pixelcount chart in FIG. 17 is 0.7257 pixel s/mOsm/L. Multiplying these twofactors together yields a calibration constant of 1.0 mOsm/L=2.25×10⁻⁵RIU. Typically, the engineering rule of thumb is that calibrationaccuracy of any measurement should be about a factor of 10 better thanthe desired accuracy required within a single measurement. Thus theabsolute calibration accuracy required to accurately measure 1.0 mOsm/Ltear osmolarity requires a calibration accuracy of the SPR device to+2.25×10^(0.6) RIU. Note that this is higher calibration accuracy thanhas been demonstrated by the reproducibility data in FIG. 13 as obtainedunder controlled laboratory conditions. This implies that reliable tearosmolarity measurements with an accuracy of ±1.0 mOsm/L may be difficultto obtain in routine practice.

FIG. 21 illustrates the relative change of the index of refraction withtemperature (i.e., Δn/Δt) for several common optical plastics. Note thatZEONEX® E48R (“E48R”), a low birefringence optical plastic manufacturedby Zeon Corporation (Japan), with an index of refraction of 1.523 in thenear infrared, is an optical polymer well suited for molding optical SPRprisms. Note that E48R has a Δn/Δt that is approximately 1.269×10⁻⁴RIU/° C. and is similar to that of the other optical plasticsillustrated in FIG. 21. As a consequence, the change in the index ofrefraction of ZEONEX® E48R per degree centigrade is approximately 28times (i.e. 1.269×10⁻⁴÷4.50×10⁻⁶) greater than the resolution requiredto accurately and repeatably measure osmolarity to ±1.0 mOsm/L. Inpractice, this implies that the temperature of the E48R SPR prism wouldhave to be either maintained within, or measured to an accuracy of about0.036° C. Either of these conditions is impractical to achieve in anordinary clinical office environment. Consequently, an extremely precisemeans of temperature calibration is required in order to achieve thedesired accuracy of the tear osmolarity measurement.

Example 5: Self-Calibrating Sensor Concept 1

A basic self-calibrating SPR sensor concept evolved from theillustrations in FIG. 22 and FIG. 23. FIG. 22 depicts a single pieceinjection molded sensor formed in an optical grade plastic. This onepiece sensor concept was intended to utilize kinematic mounting featuresto constrain it in six degrees of freedom to assure each and everysensor was precisely and repeatably aligned to the optical chassis ofthe system. As shown in FIG. 23, the concept envisioned a sensorcomprised of three segments—a base portion provides the precisionkinematic mechanical interface to the optical chassis, an SPR prismportion with a gold (or protected silver) coated SPR sensing surface formeasuring tear osmolarity, and finally, a “skirt” portion to provide thetransition between the SPR prism portion and the base portion. The prismportion provides for self-calibration by implementing means forobtaining both an optical critical angle transition and an air SPR line,preferably at two separate wavelengths of approximately 850 nm and 950nm, as well as another separate SPR line that was to appear when thegold coated sensor surface of the SPR prism was wetted by the tearfluid.

FIG. 24 illustrates a concept of an SPR sensor that uses ellipsoidalsurfaces to image light from an LED source onto the sensing surface. Asshown in FIG. 25, in order to be able to produce both an air SPR lineand a tear (or water) SPR line, there must be light incident on thesensing surface at about 42.0° to produce the air SPR line and atapproximately 64.4° to produce a tear SPR line. This is achieved byimaging light from a point source LED using an elliptical surface torelay an image of the LED onto the sensing surface (e.g., a gold coatedsensing surface) of the transparent elliptically shaped reflector. Theangles of incidence of the LED light on the internal elliptically shapedsurface are such that total internal reflectance occurs for the LEDlight. Light reflected by the gold coated SPR sensing surface is thenreflected back toward the point source LED by the left hand ellipticallyshaped inner surface and is intercepted by a beamsplitter that reflectsreturning light to an image sensor that detects the location of the SPRline. For the case of a rotationally symmetric ellipsoidal sensor, theSPR line is actually an SPR circle centered on the rotational axis ofthe ellipsoidal surface

Following the analysis of the elliptical sensor, a series of prismaticcap configurations were developed and analyzed using ZEMAX® opticaldesign software. These various configurations are illustrated in FIG. 26and FIG. 27. Generally, each of these concepts utilizes two internaltransmitting facets on the inside of the cap and either 3 or 5 externalfacets that served to totally internally reflect light along the insideof the prism portion of the cap. These sensor concepts were able toprovide images of the critical angle between air and E48R (ZEONEX® E48Rmaterial having a refractive index of approximately 1.5305), an air SPRline, and a tear SPR line. In one sensor concept, the critical angle andair SPR line are both captured in one image frame, and the tear SPR lineis captured in a subsequent image frame. In another sensor concept, allthree lines are captured in a single image frame.

Example 6: Analysis of Self-Calibrating Sensor

FIG. 28 contains a set of layout sketches for a sensor based on outputfrom the ZEMAX® optical design software. FIG. 28, Panel C, depicts aclose-up view of the sensor tip as comprised of two refracting facets(denoted by circled red numbers 1 and 7) that are disposed on aninternal surface of the sensor, four external facets that are uncoatedand reflect light via total internal reflection (denoted as surfaces 2,3, 5 and 6) and a fifth surface partially coated with a gold stripewhich is the SPR surface (denoted as surface 5 or the sensing surface).The gold coated portion of surface 5 provides the SPR line for both airand the tear osmolarity SPR measurements and the uncoated portion ofsurface 5 provides the Air critical angle transition. Both the aircritical angle transition and the air SPR line must be obtained prior tosurface 5 becoming wet by tear fluid.

The sketch in the upper left of FIG. 28 depicts the entire opticallayout of the sensor and system. Four LEDs serve as the optical sources,two operable at nominally 855 nm and two operable at nominally 950 nm.Both sets of LEDs are comprised of an 855 nm LED and a 950 nm LED, eachof which can be independently actuated. The two beams from the first setof 855 nm and 950 nm LEDs are combined into a single beam via a smalldichroic beamsplitter (not shown) so as to propagate along a common beampath as illustrated by the upper ray bundles originating from the firstset of LEDs. Considering first the case in which the 855 nm LED in thefirst set is actuated, the beam depicted as the upper bundle of rays isdirected through a window and cylinder lens and then through refractingfacet 7 toward facet 6. At facet 6 the light beam is reflected by totalinternal reflection toward facet 4, the sensor surface. The design ofthe cylinder lens is such that the light beam is nominally focused to aline on facet 4. The mid-point of the cone angle of the light incidenton the sensor surface is nominally 42 degrees, which allows acquisitionof both the air critical angle transition and the air SPR minima in asingle image frame. At the sensor surface, the light beam depicted bythe upper (light grey) ray bundle interacts with the gold and the aircontacting the gold to form an air SPR line and an air critical angletransition for the wavelength of 855 nm.

After the light beam interacts at the sensing surface the light grey raybundle light beam is reflected from facet 4 toward facet 2 at whichpoint it is totally internally reflected toward and through refractingfacet 1 and proceeds to impinge on the 2D CMOS imaging array. In thedepicted embodiment, the imaging array is a grayscale version of theAPTINA® MT9P031 1/2.5-Inch 5Mp CMOS Digital Image Sensor comprised of2592×1944 active pixels. The imager converts incident light into adigital electronic signal comprised of the digital data representing theintensity of the light at each of the 2592×1944 active pixels in theimaging array. These data can then be processed using the derivativesignal processing techniques described above to find the exact locationof the air critical angle transition and the air SPR minima angle.

Once the air critical angle transition and the air SPR minima angle aredetected on the imaging array, the 855 nm LED is deactivated and the 950nm LED is activated and a similar process is followed to acquire a setof air critical angle transition and air SPR minima angle at the 950 nmwavelength. The combination of these data comprise the automatic aircalibration sequence that occurs each time the system is brought out ofits “sleep” mode.

In a similar manner, light from the second set of 855 nm and 950 nm LEDare combined and propagated through the system along the pathillustrated by the dark gray bundle of rays shown in FIG. 28. Theprimary difference between the first set of LEDs and the second set isthat light from the second set of LEDs is totally internally reflectedby facet 5 on the way to impinging on the sensor facet and is totallyinternally reflected by facet 3 on its way to the imager. The effect ofthis difference is that the mid-point of the cone of light from thesecond set of LEDs is incident on the sensor surface at a nominal angleof approximately 64.4°. This nominal angle of incidence enablesgeneration of SPR data for liquids such as water and tear fluid. As isthe case for the first set of LEDs it is possible to obtain SPR data at855 nm and 950 nm by simply alternating the actuation of the 855 nm and950 nm LEDs.

FIG. 29, Panel A illustrates the ZEMAX® simulation of the air SPR lineand the critical angle transition using one LED from the first set ofLEDs and before surface 5 is wet with water (or tear fluid). FIG. 29,Panel B illustrates the SPR line obtained using one of the LEDs from thesecond set under the condition that surface 5 has been wet with water(or tear fluid).

Example 7: Snell's Law and Critical Angle Transition

Acquisition of accurate and precise critical angle data is an importantaspect of the calibration of the subject sensors and systems. FIG. 30illustrates the geometry of Snell's Law (the law of refraction) and thecritical angle. FIG. 30 shows the simple case of Snell's Law and thecritical angle for a single interface. A more involved optical thin filmanalysis shows that as long as the incident media has an index of n₁ andthe emergent media has an index of n₂, then the critical angle is alwaysgiven by θ_(C)=Sin−1(n₂/n_(i)), independent of the number of planeparallel layers between the incident media and the emergent media. Thusthe critical angle is invariant with respect to the materials betweenthe incident media and the emergent media—it is solely dependent on thevalues of n₁ and n₂. As a consequence, measurement of the location ofthe critical angle provides an important calibration factor for SPRmeasurements.

FIG. 31 illustrates the location of the critical angle for a gold layeron an incident medium with an index of refraction of 1.51. The emergentmedium is air with an index of refraction of 1.00027477. The goldthickness is varied from zero thickness to a thickness of 75 nm. Asshown in the chart of reflectance versus angle of incidence, thecritical angle remains stationary at 41.4757° throughout this range ofgold thickness. Since air is only weakly dispersive with respect towavelength and temperature (and its index is well characterized for bothwavelength and temperature) the primary contributor to the shift of thecritical angle will be the index of refraction of the incident media—inthe case of the subject systems, this is the index of refraction of thesensor, and any mechanical mounting tolerances of the sensor to theoptical chassis. Consequently, by making critical angle measurements at855 nm and 950 nm, and given the known and well characterized wavelengthand thermal dispersion of the ZEONEX® E48R sensor material, it ispossible to set up two equations and two unknowns to characterize themounting angle of the sensor and the index of refraction of the E48R atthe time of measurement.

Example 8: Self-Calibrating Sensor Concept 2

FIG. 32 depicts the optical layout of sensor concept 2. This concept isconsiderably simpler than sensor concept 1, utilizing two LEDs, one at855 nm and a second at 950 nm, combined into a single beam using abeamsplitter (not illustrated), a single collimating lens, a singlecylinder lens that doubles as the window for the optical chassis, asensor comprised of two internal facets and three external facets, andan image detector. Light from either the 855 nm LED or the 950 nm LEDfollow essentially the same optical path. In operation, light from theactive 855 nm LED is collimated by the collimating lens and then focusedby the cylindrical lens into a line on the sensor facet 3. After passingthrough the cylindrical lens, the beam is refracted by facet 5 acrossthe central axis of the sensor and is reflected by the uncoated facet 2.The angle of incidence of the beam on facet 2 is approximately 42.0°, sothat an air critical angle transition will be produced at this surface.The reflected beam from facet 2 is incident on the gold coated sensorfacet 3 at an angle of incidence of approximately 64.4° so as to producean SPR minimum for either water or tear fluid near the central angle ofthe focused cone of light. The thickness of the gold on facets 3 and 4is approximately 45 to 50 nm. After reflecting from the sensor surface3, the beam is incident on gold coated facet 4 and an angle of incidenceof approximately 42°, so as to produce an air SPR minimum uponreflection from this fourth facet. Finally the beam exits the sensor byrefraction through facet 1, is realigned parallel to the optical axis ofthe system by passing through the cylindrical lens, and is subsequentlyincident on the 2593×1944 pixel APTINA® imager described previously.

In a similar manner, SPR and critical angle data can be collected at the950 nm wavelength by deactivating the 855 nm LED and activating the 950nm LED. The path taken by the 950 nm light is virtually identical inthis case.

FIG. 33 illustrates a ZEMAX® simulation of the performance of sensorconcept 2, depicting its potential to produce an air critical angletransition, a tear SPR line and an air SPR line in a single image. Inprinciple, sensor concept 2 can generate a full set of air criticalangle transition, tear SPR minima and air SPR minima data within asingle captured frame, as depicted in FIG. 33.

Example 9: Analysis of Self-Calibrating Sensor Concept 1

FIG. 34 shows a further developed illustration of sensor concept 1. FIG.34 illustrates the physical size of both the LEDs and the imager asoffered mounted on a circuit board with support chips by XIMEA®. Itshould be noted that in comparison to the optical layout in FIG. 28, thelayout in FIG. 34 has been inverted top to bottom for the purpose ofproviding a more direct line of sight over the top of the system to thetip of the sensor so that the physician taking the osmolaritymeasurement can more readily place the sensing surface of the sensoronto the tear film of the eye.

Referring still to FIG. 34, light from an LED that is emitted in thegeneral direction of the sensor is collimated by the collimating lensand is focused by a cylinder lens and enters the internal hollow portionof the sensor. Inside the sensor, the light focused by the cylinder lensis refracted by the top inner facet of the sensor and is subsequentlyinternally reflected by three of the five external facets of the sensor.The second of the three facets is the sensing surface at which thecylindrically focused light comes to focus and interacts with thesensing surface gold coating and the media in contact with the externalsurface of the gold. The internal reflection by the surface followingthe sensor surface, and the subsequent refraction by the lower innerfacet primarily serve to direct the light exiting the sensor in thegeneral direction of the image sensor. An optical wedge, which may beomitted, serves to direct the axis of the exiting beam closer to thephysical axis of the system so as to lower its vertical profile.

FIG. 35 illustrates in more detail the structure for mounting the sensoron the machined aluminum optical chassis that supports the LEDs, opticalcomponents and the imager in their proper locations for creating andimaging the SPR lines and the critical angle transitions. FIG. 36illustrates the length dimension of the optical chassis and FIG. 37 andFIG. 38 illustrate the chassis vertical dimensions, and also providecomponent call-outs and more detail regarding the mounting componentsthat couple the sensor to the optical chassis.

FIG. 39 illustrates the configuration of the optical chassis whensurface mount LEDs are used. This layout also depicts a cylinder lensbonded to a plane parallel disk of optical glass which serves as awindow to prevent contaminants from entering the chain of opticalcomponents housed in the optical chassis. Bonding the cylinder lens tothe window serves to permanently set its alignment with respect to theother optical components in the chassis. FIG. 39 also shows the locationof a polarizer and its barrel. The polarizer is used to form the SPR andcritical angle transition images on the image sensor. Finally theposition of the beamsplitter that combines the light from the variousLEDs in the system is illustrated. FIG. 40 is a similar illustration ofthe chassis in a perspective view.

FIG. 41 portrays the optical and sensor chassis mounted in its exteriorhousing and also indicates the location of a control board that is usedto detect switch closures and activate the LEDs in the optical chassisin the appropriate sequence.

FIGS. 42-47 provide more detailed illustrations of the sensor. FIG. 42and FIG. 43 illustrate the three retention components that are located120° apart and upon which are three small protrusions that serve toengage a first inner surface of the bayonet mounting feature of theoptical chassis. These flexures and protrusions bias the sensor so thatthe three kinematic mount points depicted in FIG. 44 are forced intocontact with a second inner surface of the bayonet mounting feature in akinematic fashion. FIG. 45 shows an exterior end view of a sensor inaccordance with embodiments of the invention. In this illustration, theretention components no longer have a slot, which was found (using amold flow analysis software application) to cause difficulty incompletely filling the tabs during the injection molding process. FIG.46 illustrates an exterior end view of a sensor in its mating bayonetfeature of the optical chassis. FIG. 47 is a simulation of theappearance of a sensor as it would appear when molded in ZEONEX® E48Roptical polymer. The sensing surface and a plurality of facets areidentified.

Example 10: Benchtop Sensor System

FIG. 48 is an illustration of a desktop, or benchtop, system. As shownin FIG. 48, the benchtop system comprises two LED collimators, in thisexample one operational at a nominal wavelength of 855 nm and the otherat 950 nm. The LED collimators are comprised of a point source LED,followed by a circular sheet polarizer and then an appropriatecollimating lens. The depicted components are housed in brass housings.Note that the wavelengths of the collimators need not be 855 nm and 950nm, but can be any pair of wavelengths that are appropriate for thesensor and the test media being analyzed.

As shown in FIG. 48, light from the 855 nm LED collimator is incident onthe reflective hypotenuse of a 90° prism and is reflected toward thebeamsplitter. At the beamsplitter, a portion of the 855 nm beam istransmitted through the beamsplitter and subsequently through a cylinderlens and into the SPR hemi-cylinder shaped sensor and is ultimatelyfocused onto the gold coated external sensing surface of a gold coatedmicroscope slide that has been index matched to the hemi-cylinder. Theangle of incidence of this 855 nm beam on the surface of the gold is inthe range of the critical angle at 855 nm so that an 855 nm air criticalangle transition and an air SPR line can be generated. In a similarmanner, a portion of the beam from the 950 nm LED collimator can bereflected by the beamsplitter, focused by the cylinder lens, enter thehemi-cylinder and impinge on the gold coated sensing surface, also at anangle in the range of the critical angle at 950 nm so that a 950 nm aircritical angle transition and air SPR line can be generated.

In a similar fashion, the 855 nm beam that is reflected by thebeamsplitter and the 950 nm beam transmitted through the beamsplitterare combined, reflect from a second reflective hypotenuse of a 90°prism, pass through a second cylinder lens, enter the hemi-cylinder andare incident on the gold coated microscope slide at angles in the rangeof the SPR minimum and thus generate 855 nm and 950 nm SPR lines forfluids such as water solutions, tear fluids, etc.

Light reflected from the gold coated microscope slide passes through andexits the hemi-cylinder in the general direction toward the imagedetector and is analyzed by a desktop or laptop computer generally usingthe signal processing techniques described above.

FIG. 49 is a perspective view of a benchtop system without componentlabels, and FIG. 50 is a perspective view with component labels. Itshould be noted that the optical chassis of the depicted benchtop systemis formed by computer numerical control (CNC) machining its internal andexternal features from a solid billet of aluminum. This provides anextremely stable and precise optical chassis, and all criticalcomponents that require precise alignment are mounted via kinematicmounting features machined into the chassis. Consequently, there is noneed for adjustable optical mounts or other similar adjustments in orderto align the optical system. FIG. 51 is a photo of the one piece opticalchassis and its one piece CNC machined cover.

Example 11: Determination of Osmolarity of a Tear Fluid

A sensor comprising a sensing surface with a gold film was used todetermine the osmolarity of a tear fluid. The sensor was connected to asystem, and the sensing surface was contacted with air as a referencemedium. An optical signal having a wavelength of 855 nm was directed tointeract with the sensing surface at an incident angle of approximately42 degrees. The SPR signal from the sensing surface was detected using adetection component (FIG. 52, Panel A), and the pixel positioncorresponding to the minimum value of the SPR signal in air wasdetermined (FIG. 52, Panel B).

Next, a sample of tear fluid was obtained from Ursa BioScience(Abingdon, Md.) and a small volume of the sample was placed in contactwith a sensing surface of the sensor. An optical signal having awavelength of 855 nm was directed at the sensing surface at an incidentangle of approximately 64 degrees. When the tear fluid was placed incontact with the sensing surface, an instantaneous change in the pixelposition corresponding to the minimum value of the SPR signal wasdetected (FIG. 52, Panel D), relative to the pixel positioncorresponding to the minimum value of the SPR signal in air. The tearfluid was left in contact with the sensing surface for 600 seconds, anddata was collected over this time interval. The pixel positioncorresponding to the minimum value of the SPR signal changed over time,eventually reaching a plateau value. FIG. 52, Panel E, shows a graph ofthe SPR delta pixel value, measured using an optical signal having awavelength of 855 nm, and over a time interval of 60 seconds followingcontact of the sensing surface with the tear fluid. The graph in FIG.52, Panel E has y-axis units of pixels and x-axis units of seconds. Eachdata point in the depicted graph was obtained by subtracting the pixelposition corresponding to the minimum value of the SPR signal at t=0from the pixel position corresponding to the minimum value of the SPRsignal at each subsequent time point. A mathematical function wasgenerated from the plotted data points depicted in FIG. 52, Panel E, andthe function was then analyzed to determine the osmolarity of the tearfluid by comparison to a calibration data set.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples and aspects of the invention as well as specific examplesthereof, are intended to encompass both structural and functionalequivalents thereof. Additionally, it is intended that such equivalentsinclude both currently known equivalents and equivalents developed inthe future, i.e., any elements developed that perform the same function,regardless of structure. The scope of the present invention, therefore,is not intended to be limited to the exemplary aspects shown anddescribed herein. Rather, the scope and spirit of present invention isembodied by the appended claims.

1.-40. (canceled)
 41. A system comprising: (i) a sensor comprising asensing surface comprising a coated region, wherein the sensor isconfigured to: direct a first optical signal to interact with thesensing surface over a first range of incident angles; and direct asecond optical signal to interact with the sensing surface over a secondrange of incident angles, wherein the first range of incident angles isdifferent from the second range of incident angles; and (ii) an opticalchassis comprising: an optical signal generating component; a detectioncomponent; a processor; a controller; and a computer-readable mediumcomprising instructions that, when executed by the processor, cause thecontroller to: direct an optical signal having a first wavelength tointeract with the sensing surface over the first range of incidentangles to generate a first surface plasmon resonance (SPR) signal;generate an image of the first SPR signal using the detection component;determine a pixel position of a minimum value of the first SPR signal onthe generated image; direct an optical signal having a second wavelengthto interact with the sensing surface over the first range of incidentangles to generate a second SPR signal; generate an image of the secondSPR signal using the detection component; determine a pixel position ofa minimum value of the second SPR signal on the generated image; comparethe pixel position of the minimum values of the first and second SPRsignals to determine a first SPR delta pixel value; direct an opticalsignal having a first wavelength to interact with the sensing surfaceover the second range of incident angles to generate a third SPR signal;generate a series of images of the third SPR signal over a first timeinterval using the detection component; determine a series of pixelpositions that correspond to a minimum value of the third SPR signalover the first time interval; direct an optical signal having a secondwavelength to interact with the sensing surface over the second range ofincident angles to generate a fourth SPR signal; generate a series ofimages of the fourth SPR signal over a second time interval using thedetection component; determine a series of pixel positions thatcorrespond to a minimum value of the fourth SPR signal over the secondtime interval; compare a rate of change of the series of pixel positionsthat correspond to the minimum value of the third SPR signal over thefirst time interval to a rate of change of the series of pixel positionsthat correspond to the minimum value of the fourth SPR signal over thesecond time interval to determine a second SPR delta pixel value; andcompare the first and second delta pixel values to a calibration dataset.
 42. The system according to claim 41, wherein the first timeinterval and the second time interval are the same.
 43. The systemaccording to claim 41, wherein the first time interval and the secondtime interval are different.
 44. The system according to claim 41,wherein the computer-readable medium further comprises instructionsthat, when executed by the processor, cause the controller to compare apixel position that corresponds to a minimum value of at least one ofthe SPR signals to a reference feature.
 45. The system of claim 44,wherein the reference feature comprises a pixel position of a maximumvalue of a critical angle signal.
 46. The system according to claim 45,wherein the sensor comprises a coated region and a non-coated region,and wherein the critical angle signal is generated from the non-coatedregion.
 47. The system of claim 44, wherein the reference featurecomprises a pixel position of an opto-mechanical reference (OMR). 48.The system according to claim 41, wherein the first range of incidentangles spans 40 to 45 degrees.
 49. The system according to claim 41,wherein the sensor is configured to direct the first optical signal tointeract with the sensing surface at a first incident angle of 42degrees.
 50. The system according to claim 41, wherein the second rangeof incident angles spans 62 to 67 degrees.
 51. The system according toclaim 41, wherein the sensor is configured to direct the second opticalsignal to interact with the sensing surface at a second incident angleof 64 degrees.
 52. The system according to claim 41, wherein the firstwavelength is 855 nm and the second wavelength is 950 nm.
 53. The systemaccording to claim 41, wherein the sensor is configured to be removablycoupled to the optical chassis.
 54. A method for determining theosmolarity of a sample, the method comprising: contacting a sensingsurface of a system according to claim 1 with a reference medium;directing an optical signal having a first wavelength to interact withthe sensing surface over the first range of incident angles to generatea first surface plasmon resonance (SPR) signal; generating an image ofthe first SPR signal using the detection component; determining a pixelposition of a minimum value of the first SPR signal on the generatedimage; directing an optical signal having a second wavelength tointeract with the sensing surface over the first range of incidentangles to generate a second SPR signal; generating an image of thesecond SPR signal using the detection component; determining a pixelposition of a minimum value of the second SPR signal on the generatedimage; comparing the pixel position of the minimum value of the firstand second SPR signals to determine a first SPR delta pixel value;contacting the sensing surface with a test medium; directing an opticalsignal having a first wavelength to interact with the sensing surfaceover the second range of incident angles to generate a third SPR signal;generating a series of images of the third SPR signal over a first timeinterval using the detection component; determining a series of pixelpositions that correspond to a minimum value of the third SPR signalover the first time interval; directing an optical signal having asecond wavelength to interact with the sensing surface over the secondrange of incident angles to generate a fourth SPR signal; generating aseries of images of the fourth SPR signal over a second time intervalusing the detection component; determining a series of pixel positionsthat correspond to a minimum value of the fourth SPR signal over thesecond time interval; comparing a rate of change of the series of pixelpositions that correspond to the minimum value of the third SPR signalover the first time interval to a rate of change of the series of pixelpositions that correspond to the minimum value of the fourth SPR signalover the second time interval to determine a second SPR delta pixelvalue; and comparing the first and second delta pixel values to acalibration data set to determine the osmolarity of the sample.
 55. Themethod according to claim 54, wherein the first time interval and thesecond time interval are the same.
 56. The method according to claim 54,wherein the first time interval and the second time interval aredifferent.
 57. The method according to claim 54, further comprisingcomparing a pixel position that corresponds to a minimum value of atleast one of the SPR signals to a reference feature.
 58. The method ofclaim 57, wherein the reference feature comprises a pixel position of amaximum value of a critical angle signal.
 59. The method of claim 57,wherein the reference feature comprises a pixel position of anopto-mechanical reference (OMR).
 60. The method according to claim 54,wherein the first wavelength is 855 nm and the second wavelength is 950nm.
 61. The method according to claim 54, wherein the reference mediumis air.
 62. The method according to claim 54, wherein the sample is abiological sample.
 63. The method according to claim 62, wherein thebiological sample is a tear fluid.
 64. The method according to claim 54,wherein the first time interval ranges from 0.001 seconds to 90 seconds.65. The method according to claim 54, wherein the second time intervalranges from 0.001 seconds to 90 seconds.